Biodegradable Polymeric Nano formulation of Acyclovir and Acyclovir prodrug for Enhanced Therapeutic Efficacy Against Herpes Simplex Virus Infections

Biodegradable Polymeric Nano formulation of Acyclovir and Acyclovir prodrug for Enhanced Therapeutic Efficacy Against Herpes Simplex Virus 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 Article Biodegradable Polymeric Nano formulation of Acyclovir and Acyclovir prodrug for Enhanced Therapeutic Efficacy Against Herpes Simplex Virus Infections Sushruta S Hakkimane, Divakarareddy Vemanna Paladugulu, Santosh L Gaonkar, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7427697/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Herpes simplex virus-1 (HSV-1) is a widespread, recurrent infection that causes herpes simplex keratitis (HSK), an ocular disease that can lead to vision impairment. Conventional acyclovir (ACY) therapy is limited by poor bioavailability, rapid clearance, and frequent dosing. We developed an esterified acyclovir prodrug (ACPD) formulated as biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) to improve stability, enable sustained release, and enhance antiviral activity against HSV-1. ACPD was synthesized and characterized by FTIR, NMR, and mass spectrometry. Its increased hydrophobicity improved encapsulation in PLGA relative to ACY. ACY-NPs and ACPD-NPs were prepared by nanoprecipitation and single-emulsion solvent evaporation method, respectively. Optimized NPs measured 200–300 nm with polydispersity indices of 0.23–0.47 and zeta potentials of − 27 to − 29 mV; SEM showed uniform particles. ACPD-NPs provided sustained release for 28 days. Cytotoxicity in Vero cells yielded CC50 values of 960 µM (ACY-NPs) and 1000 µM (ACPD-NPs). In HSV-1-infected Vero cells, ACY-NPs exhibited greater antiviral efficacy than free ACY, whereas ACPD and ACPD-NPs showed minimal plaque reduction because the in vitro media lacked esterase to convert ACPD to ACY; with esterase, cleavage was confirmed by RP-HPLC. Corneal tissues contain esterase, in vivo activation may improve ACPD efficacy and bioavailability and overall therapeutic performance. Biological sciences/Biotechnology Biological sciences/Drug discovery Biological sciences/Microbiology Physical sciences/Nanoscience and technology Acyclovir Acyclovir prodrug nanoparticles poly (lactic-co-glycolic acid) Herpes simplex virus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Herpes simplex virus (HSV) is a common viral infection that mainly causes oral herpes, genital herpes and ocular infections like keratitis. HSV infections are widespread, posing a significant clinical challenge worldwide. HSV-1 and HSV-2 infections affect over two-thirds of the global population, with rising incidences. As per the WHO estimates in 2020, HSV-1 had infected 3.8 billion people under the age of 50[ 1 ]. Around 1.8 million people were affected with herpes simplex keratitis (HSK), caused by HSV-1 and leads to corneal inflammation, scarring and vision impairment[ 2 ]. Herpes simplex keratitis (HSK) is one of the leading causes of infectious blindness. As HSV-1 undergoes the latency stage after a primary infection, it enters and hides in sensory ganglia[ 3 ]. Certain factors like surgery, stress, and immunosuppression can reactivate the virus, causing the recurrence of infection, which leads to vision impairment and vision loss[ 4 ]. To inhibit HSV-1, antiviral drugs like acyclovir, valacyclovir, and famciclovir are used. These are delivered orally or topically and often face challenges like poor drug delivery due to barriers, latency, poor bioavailability, premature degradation, short half-lives, and low penetrating capacity to certain cellular sites[ 5 – 7 ]. These challenges pose a threat of recurrence and cause corneal damage in HSK patients[ 8 ]. The oral treatment may extend up to 6 to 9 months, leading to various side effects and drug resistance. Despite ongoing efforts, no approved vaccines are available yet, though several candidates show evident results in early clinical trials [ 9 ]. Several barriers and several factors hamper the rapid advancement of antiviral drugs. There is a constant need for the development of novel medications and the enhancement of existing drug formulations. Among the currently available antiviral agents, acyclovir (ACY) remains the most widely used treatment for HSV-related infections. Acyclovir (ACY) is one of the first-line treatments for HSV-1. ACY is a synthetic purine nucleoside analogue with the chemical formula C₈H₁₁N₅O₃ and a molecular weight of 225.20 g/mol (Fig. 1 ). The Food Drug Administration (FDA) has approved it for treating genital herpes and HSV encephalitis due to its affordability, safety, and tolerability[ 10 ]. ACY requires intracellular activation to exert its antiviral effects. Viral thymidine kinase (TK, UL23 gene) catalyses the conversion of ACY into ACY monophosphate, which is further phosphorylated to form ACY triphosphate. This process increases ACY concentration in infected cells while reducing cellular efflux, thereby enhancing its antiviral activity[ 11 , 12 ]. ACY effectively inhibits viral DNA replication and synthesis. Although it reduces symptoms and recurrences, ACY does not eliminate the virus[ 13 ]. Though there are many topical ACY formulations, they fail to reach the infected site due to various physiological factors. As ACY is hydrophilic, it cannot pass through the lipophilic barriers to reach HSV-1[ 14 ]. Even the topical creams and gels have poor retention, leading to short exposure time[ 15 ]. ACY has low bioavailability; only a small fraction will reach infected cells. ACY will fail to reach the latent virus as it does not penetrate through the deeper layer of tissue and neurons. Due to a shorter half-life, ACY is rapidly eliminated, so frequent dosing is needed. To overcome ACY’s limitations, the derivatives, such as valacyclovir (VACV), the L-valyl ester of ACY, have been developed to improve bioavailability following oral administration[ 16 ]. Even though they require fewer doses compared to acyclovir, the higher systemic levels will cause renal toxicity and other side effects, like headache, and they also require a long-term treatment. To overcome these limitations, we are aiming to develop esterified ACPD, which serves as proof of concept to treat herpes simplex keratitis (HSK) caused by HSV-1. In the cornea of the eye, there are esterase enzymes that help to cleave ACPD to ACY[ 17 – 19 ] and ACPD will be converted to an active form only at the site of the action. This will increase the stability and the maximum amount of drug will reach the infected site. Even though there are wide treatment strategies to treat herpetic eye diseases, no intracameral formulation is available for the treatment of stromal and endothelial infected herpetic eye disease. Therefore, the intracameral formulations like anterior Chamber (AC) injection with ACY or ACPD NPs or implant with sustained release directly at the local tissue can reduce systemic side effects and increases the effectiveness of treating HSV-1[ 20 – 22 ]. Nanotechnology-driven formulations present a novel approach for improving the localised efficacy of antiviral agents and advancing therapeutic strategies for viral infections. These formulations improve drug solubility, protect the drug from degradation, and help overcome biological barriers. Additionally, they enable controlled and sustained drug release at the site of infection, enhancing therapeutic outcomes[ 21 , 23 , 24 ]. As reported in the literature, numerous attempts have been made to alter the pharmacokinetics and improve bioavailability of antiviral medications by making it as prodrugs. Prodrugs also improve the physicochemical characteristics like solubility, bioavailability and stability, which creates more efficient approach of treating viral diseases by overcoming the limitations of traditional antiviral medications[ 23 ]. A combination of prodrugs and nanotechnology will be a better strategy to treat HSV-1. Nano-formulations are a potential strategy to enhance the therapeutic efficacy by creating controlled drug delivery using nanoparticulate carriers. Our previous work from the lab supports prodrugs are more stable in different pH conditions and they are as effective as pure drug at the same molar concentration. Drug and prodrug encapsulated in NPs are more effective in suppressing the growth of tuberculosis strain at much lower concentrations compared to pure drug [ 25 ]. The findings encouraged us to adapt the similar strategy to improve the effective treatment for the herpes virus infections. The combination of nano-formulations with prodrug will yield better efficacy compared to individual treatment strategies. In this study, prodrug of ACY is prepared by reacting benzoic acid with ACY which creates hydrolytically cleavable ester bond with ACY, referred to as ACPD in this manuscript. ACPD-loaded PLGA NPs were prepared, which increases the stability and its encapsulation in NPs. Further, assessed their cytotoxicity and plaque reduction activity compared to pure ACY and ACY NPs. We also conducted ACPD in vitro studies with calculated amount of esterase enzyme in buffer. the results show cleavage of ACPD to ACY and it is measured and quantified using RP-HPLC. MATERIALS AND METHODS All reagents were obtained from commercial suppliers and used without further purification. The infrared spectrum of ACPD was recorded using a Shimadzu FTIR spectrophotometer. The 1 H NMR (400 MHz) and 13 C NMR (100 MHz) spectra were acquired on a Bruker AM 400 MHz NMR spectrometer with TMS as an internal standard. High-resolution mass spectrometry (HRMS) analysis was performed via the time of flight (TOF) and electrospray ionization (ESI) method. Reaction progress and completion were monitored by thin layer chromatography (TLC) on silica gel plates, with spot visualization under UV light (365 nm). The melting points of the compounds were determined using the open capillary method. Poly(D, L-lactide-co-glycolide) (PLGA 50:50; molecular weight: 100,000–120,000 Da) was purchased from Durect Corporation (Birmingham, AL, USA). Acetonitrile and methanol were obtained from Merck Millipore, India. Acyclovir (ACY) was kindly gifted by Zydus Lifesciences Ltd. (Ahmedabad, India). Dimethyl sulfoxide (DMSO), 4-dimethylaminopyridine (DMAP), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were purchased from Spectrochem Pvt. Ltd. (Mumbai, India). Benzoic acid and TLC plates were obtained from Merck Millipore, India. All other reagents and solvents used were of HPLC grade. Polyvinyl alcohol (PVA, 87–89% hydrolyzed; molecular weight: ~120,000 Da) was purchased from Thomas Baker (Mumbai, India). Cell culture reagents included Minimal Essential Medium (MEM), 96-well plates, and 12-well plates from HiMedia (Thane, India), and fetal bovine serum (FBS) from Gibco (Mumbai, India). Trypsin, sterile phosphate-buffered saline (PBS), and Trypan Blue were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Vero cells (ATCC® CCL-81™), procured from the American Type Culture Collection (ATCC) and the HSV-1 MacIntyre strain (ATCC® VR-539™) were used in antiviral studies. Agarose overlay medium and esterase from porcine liver (molecular weight: 168 kDa) were procured from Sigma-Aldrich, Merck (Darmstadt, Germany). Chemistry Synthesis of 2-((2-amino-6-oxo-3,6-dihydro-9 H -purin-9-yl)methoxy)ethyl benzoate (ACPD)(3) An oven-dried, 50 mL round-bottom flask equipped with a magnetic stir bar was charged with ACY (0.44 mmol), benzoic acid (0.44 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 0.55 mmol), and 4-dimethylaminopyridine (DMAP, 0.46 mmol), serving as a coupling reagent and catalyst, respectively. To this, 2 ml of anhydrous DMF was added under nitrogen environment to maintain the anhydrous conditions. The reaction mixture was stirred at room temperature overnight. The reaction progress was monitored by TLC using a methanol: chloroform (1:9) solvent system. Upon completion, the reaction mixture was quenched with cold water, resulting in the formation of a precipitate. The precipitate was filtered, dried, and subsequently recrystallised from isopropanol. Spectral Data: 2-((2-amino-6-oxo-3,6-dihydro-9 H -purin-9-yl)methoxy)ethyl benzoate (3) White solid, yield = 88%, MP = 227–229°C, FTIR (cm − 1 ): 3447 & 3319 (NH 2 stretching), 3194 (NH stretching), 3101 (CH stretching aromatic), 2928 (CH stretching aliphatic), 1713 (C = O ester), 1687 (C = O pyrimidine),1604 (N = C stretching), 1572 (C = C stretching), 1386 (CH 2 bending), 1277 (C-O-C stretching). 1 H NMR (400 MHz, DMSO) δ 10.63 (s, 1H), 7.87 (d, J = 7.2 Hz, 3H), 7.66 (d, J = 7.2 Hz, 1H), 7.52 (t, J = 7.7 Hz, 2H), 6.51 (s, 2H), 5.41 (s, 2H), 4.36 (s, 2H), 3.82 (d, J = 5.2 Hz, 2H). 13 C NMR (100 MHz, DMSO) δ 166.08, 157.27, 154.37, 151.91, 138.21, 133.84, 129.94, 129.57, 129.20, 117.05, 72.35, 66.92, 64.02. HRMS (TOF): 330.16 (M + H) + , 330.12 (Calculated M + H); MF: C 15 H 15 N 5 O 4 . Quantitative Analysis by Liquid Chromatography Optimisation and Method Development for ACY and ACPD The analysis was performed on a reverse-phase high-performance liquid chromatography (RP-HPLC) system (Shimadzu LC-20AD), integrated with an autosampler (SIL-20AC), a PDA detector (SPD-M40). Chromatographic separation was performed using a Phenomenex® Luna C18 column (5 µm particle size, 100 Å pore diameter, 250 mm length), followed by data acquisition and processing using LabSolutions software (Shimadzu, Kyoto, Japan). Binary gradient mode with an injection volume of 20 µL was used. All solvents and reagents employed were of HPLC grade to ensure analytical precision. For ACY analysis, the mobile phase was a mixture of 10:90 acetonitrile and water, with a flow rate of 1 mL/min, and a run time of 10 minutes, with detection performed at 254 nm. A stock solution (1 mg/mL) was prepared by dissolving 10 mg of the drug in 10 mL of water, and a calibration curve was constructed using different concentrations. ACPD was analysed using a reverse-phase HPLC method with a binary gradient mobile phase of acetonitrile and water. The flow rate was maintained at 1.0 mL/min, and the total run time was 16 minutes. The gradient flow started with 20% acetonitrile for 1 minute, increased to 70% by 5 minutes, then reduced back to 20% by 8 minutes, and held at 20% until 16 minutes for system stabilisation. Detection was carried out at 245 nm. A stock solution of ACPD (1 mg/mL) was prepared by dissolving 10 mg of the compound in 10 mL of acetonitrile containing 1% DMSO. A calibration curve was constructed using serial dilutions of this stock. The same method was used for the simultaneous estimation of ACPD and ACY. Method validation Following system equilibration, three blank injections were performed to ensure baseline stability. System suitability was confirmed by injecting five replicates of a calibration standard solution of ACY. Method development and validation were conducted as per established procedures [ 26 , 27 ]. The analysis was performed with an injection volume of 20 µL, a total run time of 10 minutes, and detection at 254 nm. This method was successfully used to analyse ACY in nanoparticle drug formulations. Validation parameters such as linearity, precision, and repeatability were evaluated as per ICH guideline Q2(R1) (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use). Repeatability was checked using intraday variation, while intermediate precision was assessed through inter-day variation. Overall precision included repeatability, intermediate precision, and reproducibility. Preparation of ACPD and ACY loaded nanoparticles Single Emulsion solvent evaporation Technique to make ACPD NPs ACPD-loaded PLGA nanoparticles were prepared by the single emulsion solvent evaporation method. PLGA and ACPD (2:1, w/w) were dissolved in chloroform to form the organic phase, which was stirred (SCHOTT Instruments, Mainz, Germany) and added dropwise into 12 mL of 2.5% (w/v) PVA solution kept in an ice bath. The mixture was sonicated using a probe sonicator (QSonica CL-334, Newtown, CT, USA) at 40% amplitude for 6 minutes to form an oil-in-water emulsion. The emulsion was stirred for 12 hours at room temperature to evaporate the organic solvent. Nanoparticles were collected by centrifugation (Eppendorf 5430R, Hamburg, Germany) at 11,000 rpm for 30 minutes at 4°C. The pellet was washed, redispersed in deionised water, and lyophilised (LaboGene ScanVac CoolSafe, Lynge, Denmark) at − 108°C. The dried nanoparticles were stored at − 20°C. Blank nanoparticles (without drug) were prepared using the same procedure, with only PLGA in chloroform [ 25 ]. Nanoprecipitation technique to make ACY NPs For the preparation of the ACY NPS, the nanoprecipitation technique was used to enhance the drug loading. In 10 mL acetone, an organic solution of a biodegradable polymer 1:1 PLGA and ACY were made. Dropwise, the organic phase was added to a 30 ml filtered pure water under continuous magnetic stirring. Centrifugation at 11,000 rpm at 4°C for 30 minutes was used to separate the non-encapsulated ACY. The supernatant was discarded. The final pellet was dispersed in 5 mL of water and lyophilised to get nanoparticles. Nanoparticles were stored at -20 °c for further use[ 28 ]. Characterisation of nanoparticle formulations Particle size Surface charge and Surface morphology Particle size analyser (Nano-ZS, Malvern, Westborough, United States) was used to determine the average diameter of the particles, size distribution, polydispersity index, and Zeta potential of the prepared formulations. Nanoparticles were suspended in deionised water and bath sonicated for 30 seconds before measurement. Electrode was used to measure the zeta potential on the same instrument. The size and shape of ACPD and ACY NPs were analysed using scanning electron microscope (7610FPLUS, Jeol, Japan). Images were captured at an accelerating voltage of 5 kV, with a working distance of 7 mm and a magnification of 10,000x. Determination of ACY and ACPD encapsulation in nanoparticles The drug loading in PLGA nanoparticles (NPs) was determined by quantifying the amount of encapsulated drug per milligram of nanoparticles. A fixed quantity of freeze-dried NPs were accurately weighed and suspended in a measured volume of methanol. The suspension was kept on a rocker shaker (REMI, Mumbai, India) for 48 hours to facilitate drug release from the polymeric matrix. After incubation, the samples were centrifuged at 11,000 rpm for 20 minutes, and the resulting supernatant was collected and evaporated to dryness. The dried residue was reconstituted in an appropriate solvent and analysed using RP-HPLC to quantify the encapsulated drug. All measurements were performed in triplicate, and the mean value was reported. Stability Study of ACY and ACPD The stability of ACY and ACPD was evaluated in phosphate buffer solutions of pH 4.5, 5.2, and 7.4. Known amount of each drug was weighed and diluted in the respective buffers. Each sample, prepared in triplicate, was analysed by RP-HPLC to record initial readings. The drug-containing samples were incubated at 37°C on a rotary shaker, and at defined time intervals, they were transferred into individually labelled HPLC vials for analysis. The study was conducted over 15 days, and the graph was plotted for percentage of stable drug in Y axis and days in X axis to quantify the degradation amount of ACY and ACPD. In Vitro Drug Release Kinetics In vitro drug release studies were conducted to evaluate the release of ACY from ACY-PLGA NPs and ACPD from ACPD-PLGA NPs. A known amount of lyophilised nanoparticles were accurately weighed and suspended in buffer solutions of pH 4.5 and 7.4. The samples prepared were in triplicate, incubated at 37°C on a rocker shaker to simulate physiological conditions. At defined time intervals, the suspensions were centrifuged at 11,000 rpm for 20 minutes, and the supernatant was collected and analysed using RP-HPLC to quantify the released drug. The nanoparticles pellet was resuspended in a fixed volume of fresh buffer and returned to the shaker for continued incubation. This process was repeated until maximum drug release was achieved. Drug release kinetics were evaluated by calculating the cumulative percentage of drug released over time (in days). Cytotoxicity profile of test compounds in Vero cells (CPE-based) Vero cells were maintained in MEM supplemented with 10% FBS and gentamycin (40 mg/mL). The cells (1.5 × 10⁴ cells/well) were seeded in a 96-well microtiter plate and incubated at 37°C in a humidified 5% CO₂ environment. Test compounds were assessed at concentrations ranging from 1000 to 1.9 µM for cytotoxicity. The test compounds included ACY, Acyclovir-loaded nanoparticles (ACY- NPs), ACPD and ACPD loaded NPs. A stock solution of 2 mM was prepared for each test compound, followed by serial dilutions to achieve the final concentrations of 1000 µM, 500 µM, 125 µM, 62.5 µM, 31.2 µM, 15.6 µM, 7.8 µM, 3.9 µM, and 1.9 µM. These dilutions were prepared in MEM supplemented with 2% FBS. Each test compound concentration was added in triplicate to the Vero cell monolayer. The cells were incubated for 48 h at 37°C in a CO 2 incubator. Cytotoxicity was evaluated by monitoring changes in cell morphology and growth rate in cells treated with the test compounds, compared to untreated control cells. The cells were observed under inverted microscope for any morphological changes such as deterioration and detachment of cell monolayer. The plates were incubated for 48 hrs and then fixed with 70% v/v methanol and stained with 0.5% w/v crystal violet. Absorbance was measured at 490 nm using an ELISA reader (BioTek, ELx800, USA). The percentage of cytotoxicity was determined using the formula: Percentage of cell cytotoxicity = [A C – A T / A C ] *100; where A C = Absorbance of cell control; and A T = Absorbance of test wells. Virus plaque reduction assay The assay was performed using the non-cytotoxic concentrations (2-160 µM) of the test compounds. The dilutions of the test compounds were prepared in MEM MM. A concentration of 100 PFU (Plaque forming unit) of HSV-1 was used for the assay. A confluent monolayer of Vero cells (1.25*10⁵ cells/ml) in 12-well plates were used for the assay. Media was discarded and 200 µl of fresh media (MEM MM) was added to all wells. The cells were inoculated with 100 PFU of HSV-1. After 1 hour of incubation at 37°C, virus was removed and 100 µL of different concentrations of the test compounds were added to the respective wells. 100 µL MEM MM was added to the cell and virus control wells, followed by the addition of agarose overlay media to all the wells. Post 24 hours of incubation, a second agarose overlay media with 0.3% v/v neutral red satin was added to visualize the plaques. Post 14 hours of incubation, number of plaques in each treatment group were counted using light box. Plaques were represented as mean ± SD and analyzed statistically in comparison to virus control. Percentage reduction in viral plaques was calculated using the formula: Percentage reduction in viral plaques = [(Average number of plaques in virus control - Average number of plaques in test) / Average number of plaques in virus control] *100 Enzymatic cleavage of ACPD to ACY For enzymatic cleavage studies, 15 units of porcine liver esterase were dissolved in 1 mL of phosphate buffer (pH 8) to prepare the enzyme stock solution. From this, 10 µL (equivalent to 0.15 units) was added to ACPD solution, which was also prepared in phosphate buffer (pH 8) [ 19 , 29 ]. Prior to enzyme addition, ACPD solution was analysed using the previously optimised RP-HPLC method developed for the quantification of ACPD. After confirming the retention time of ACPD, the enzyme was added, and the reaction mixture was incubated at 37°C. At 15-minute intervals, samples were analysed by RP-HPLC to monitor the enzymatic breakdown of ACPD into ACY. RESULTS AND DISCUSSION Chemistry ACY was reacted with benzoic acid to selectively synthesize 2-((2-amino-6-oxo-3,6-dihydro-9 H -purin-9-yl)methoxy)ethyl benzoate (ACPD) via O -benzoylation, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as a coupling agent (Fig. 2 ). The structure of the resulting product was confirmed using FTIR, 1 H NMR, 13 C NMR, and High-Resolution Mass Spectrometry (HRMS). The FTIR spectra of ACPD derivative exhibited characteristic absorption bands at 3447 cm − 1 and 3319 cm − 1 corresponding to asymmetric and symmetric NH 2 stretching vibrations. A peak at 3194 cm − 1 was attributed to NH stretching. The carbonyl absorptions appeared at 1713 cm − 1 (C = O ester) and 1687 cm − 1 (C = O pyrimidine). Additionally, the N = C stretching vibration was observed at 1604 cm − 1 (Fig. 3 a). The Individual FTIR spectrum of ACY and ACPD are given in Supplementary Fig. S1 and S2. This reaction selectively yielded the O -benzoylated ACY, where the hydroxyl group of ACY underwent benzoylation while the primary amine (NH 2 ) on the purine ring and the secondary amine (NH) on the pyrimidine ring remained unaffected. This selectivity was confirmed by 1 H NMR, which showed the disappearance of the hydroxyl (OH) proton signal between 4.40 and 5.00 ppm in the product, while the amine signals were preserved at 6.51 ppm and 10.62 ppm, respectively (Fig. 3 b). The 1 H NMR spectrum of ACPD 3 further exhibited distinct signals for the three methylene groups at 3.82, 4.35, and 5.41 ppm, while the signal for the free primary amine appeared at 6.5 ppm. Aromatic protons, including five from the phenyl ring and one from the purine ring, resonated between 7.49 and 7.90 ppm. The purine CH proton overlapped with the phenyl protons ortho to the carbonyl group at 7.81–7.87 ppm. A distinct signal for the purine NH proton appeared at 10.62 ppm. The disappearance of the hydroxyl signal around 4.7 ppm, along with the emergence of five additional aromatic proton signals, confirmed the successful O -benzoylation of ACY. The 1 H NMR spectrum of ACY and ACPD are given in Supplementary Fig. S3 and S4. The 13 C NMR spectrum displayed 13 peaks consistent with the expected structure. The highly deshielded benzoyl carbonyl carbon appeared at 166.06 ppm, while the three aliphatic methylene carbons resonated at 72.35, 66.92, and 64.02 ppm. The remaining carbon signals were observed between 117.05 and 116.09 ppm (Fig. 3 c). Further, mass spectrometry was done to validate mass of the synthesised derivatives, which was found to match with theoretical value (Supplementary Fig. S5). RP-HPLC for Quantitative Analysis The developed RP-HPLC method enabled the accurate and reproducible quantification of ACY and ACPD with sharp, well-resolved peaks and minimal tailing. ACY was eluted at 3.865 minutes and detected at 254 nm (Fig. 4 a), while ACPD showed a retention time of 8.477 minutes, detected at 245 nm (Fig. 4 b). Simultaneous estimation of both analytes revealed retention times of 2.767 min for ACY and 8.948 min for ACPD (Fig. 4 c). Linearity was established across a broad concentration range for both drugs. The calibration curve for ACY (0.976–200 µg/mL) showed coefficient of determination (R²) of 0.9966 with the regression equation y = 59045x (Supplementary Fig. S6), while ACPD exhibited strong linearity with y = 31734x and R² = 0.9986 (Supplementary Fig. S9). percent relative standard deviation (%RSD) values for both peak area and retention time remained below 1% and 0.5%, respectively, indicating excellent precision, reproducibility, and system suitability. The stacked chromatograms for ACY and ACPD are provided in Supplementary Fig. S7 and Fig. S10, respectively. Precision plots and data are included in Supplementary Table S1 and Fig. S8. The method was validated as per ICH guideline Q2(R1), confirming its robustness in terms of linearity, accuracy, and precision [ 30 ]. The validated RP-HPLC method offers several advantages over traditional methods, including shorter analysis times, enhanced resolution, and better adaptability to complex matrices [ 31 ]. Preparation and characterization of ACY and ACPD loaded nanoparticles Size, Shape and surface morphology The prepared ACY-PLGA NPs had a mean particle size of 211 ± 24 nm with PDI 0.477 and a mean zeta potential of -27 ± 4 mV (Fig. 5 a and 5 b). ACPD-PLGA NPs had a mean particle size of 293 ± 24 nm with PDI 0.230 and a mean zeta potential of -29 ± 4 mV. (Fig. 5 c and 5 d). The average particle size of drug-free PLGA nanoparticles was found to be 249 ± 43 nm and mean Zeta potential − 49 ± 10mV as shown in Supplementary Fig. S11 and Fig. S12. SEM images revealed the size, shape and surface morphology of the prepared NPs (Fig. 5 e). Images confirmed that the particles are spherical and are evenly distributed. The particle size measurements aligned with the results obtained from the particle size analyser, and no cracks or pores were observed, confirming the smooth surface morphology of the prepared nanoparticles. This study aimed to enhance the stability and bioavailability of ACY by encapsulating it within FDA-approved, biodegradable PLGA nanoparticles. Nanoparticles were prepared using two widely used methods. ACPD NPs and blank NPs were synthesized using the optimized single emulsion and solvent evaporation method, as described previously[ 32 , 33 ]. The method is typically suitable for hydrophobic drugs. However, due to the hydrophilic nature of ACY, encapsulation efficiency was suboptimal when the double emulsion solvent evaporation method was used. Consequently, the nanoprecipitation technique was adopted, which significantly improved ACY encapsulation within the nanoparticles. This highlights the importance of selecting suitable nanoformulation techniques based on the drug's physicochemical properties to ensure optimal nanoparticle performance. In our previous work, nanoparticle preparation was optimised by varying sonication time, PVA concentration, and drug amount as key formulation parameters[ 34 ]. PLGA degrades into non-toxic lactic and glycolic acid monomers. Polyvinyl alcohol (PVA) served as a surfactant, facilitating emulsification for encapsulation of drug into NPs. Excess PVA was removed through repeated resuspension and centrifugation in deionized water (3× at 11,000 rpm). Various PVA concentrations (1%, 2.5%, and 5%) were tested, but 2.5% gives better particle size, stability and reduces coalescence of the particles[ 25 , 34 ]. Zeta potential and particle size were measured using a Zetasizer. Zeta potential, a key indicator of colloidal stability, reflects the electrostatic repulsion between particles. Values exceeding ± 30 mV typically indicate stable systems due to sufficient charge repulsion, which prevents aggregation and promotes redispersion[ 35 ]. The obtained values confirmed the physical stability of the optimized nanoparticle formulations. Drug loading into PLGA NPs The amount of drug loaded in NPs was quantified by measuring the drug encapsulated in micrograms per milligram of nanoparticles (NPs). The optimised PLGA-ACY NPs exhibited an encapsulation of 186 ± 16 µg/mg, while the PLGA-ACPD NPs showed a higher encapsulation amount of 271 ± 12 µg/mg. Stability Analysis of ACY and ACPD The stability of ACY and ACPD was evaluated in phosphate buffer solutions at pH 7.4, 5.2, and 4.5 to mimic physiological conditions, where pH 7.4 represents blood plasma, pH 5.2 simulates the endosomal environment, and pH 4.5 corresponds to lysosomal conditions. Drug solutions were incubated at 37°C and analysed by RP-HPLC over 15 days. As shown in Fig. 6 a, ACY is quite stable, approximately 96% of the drug remained at pH 7.4 after Day 15. At lower pH 5.2 (89%) and pH 4.5 (85%), the stability of ACY decreases. These findings align with previous reports on the stability of ACY, which showed significant degradation under acidic and oxidative conditions, while only minor degradation occurred in neutral and alkaline environment [ 36 ]. In contrast, Fig. 6 b shows that ACPD maintained greater stability across all pH conditions, retaining 93–98% of its initial concentration over the same period. These results suggest that ACPD is more stable than ACY in both neutral and acidic conditions. In Vitro Drug Release Kinetics The drug release profiles of ACY- and ACPD-loaded PLGA nanoparticles were evaluated at pH 4.5 and 7.4 to simulate lysosomal and plasma environments, respectively. ACY-loaded NPs showed a rapid initial burst, releasing approximately 68% at pH 4.5 and 73% at pH 7.4 within the first 6 hours. By 24 hours, the cumulative release reached 70% at pH 4.5 and 74% at pH 7.4, after which it remained nearly constant over the 8-day study period(Fig. 7 a). In contrast, ACPD-loaded NPs exhibited a more gradual and sustained release, with 16% at pH 4.5 and 20% at pH 7.4 in the first 6 hours, followed by 35% at pH 4.5 and 42% at pH 7.4 by 24 hours. The cumulative release extended to 86% at pH 4.5 and 91% at pH 7.4 over 28 days(Fig. 7 b). The faster release of ACY can be attributed to its hydrophilic nature, which facilitates rapid diffusion through the aqueous polymer matrix [ 37 ]. In contrast, the slower release of ACPD reflects its hydrophobicity and stronger interaction with the PLGA network. PLGA nanoparticles, owing to their biocompatibility and biodegradability, provide a protective environment and supports controlled and sustained drug release while minimising premature degradation [ 38 , 39 ]. ACPD, synthesised from ACY via ester bond formation, undergoes hydrolytic cleavage to release ACY under acidic conditions more than at neutral pH. In vitro studies of ACPD-loaded PLGA nanoparticles revealed both sustained release of ACPD and its hydrolytic cleavage to ACY over time. The formation of ACY from ACPD was confirmed by the RP-HPLC method developed specifically for ACPD quantification. The cleavage was more evident at pH 4.5 than at pH 7.4, consistent with proton-catalysed hydrolysis of the O-benzoyl ester bond under acidic conditions [ 40 ]. RP-HPLC chromatograms confirming ACY release from ACPD are provided in Supplementary Fig. S13. Cytotoxicity profile of test compounds in Vero cells The cytotoxicity profiles of ACY, ACPD, ACY- NPs, and ACPD- NPs were assessed in Vero cells using crystal violet staining. The 50% cytotoxic concentration (CC50) values were determined to be 1230 µM for ACY, 960 µM for ACY-NPs, 800 µM for ACPD, and 1000 µM for ACPD-NPs, as illustrated in Fig. 8 a. All nanoparticle formulations exhibited no cytotoxicity up to a concentration of 160 µM, while ACY and ACPD demonstrated minimal cytotoxic effects. The cytotoxicity ACY is given in Supplementary Fig. S14. Antiviral activity by plaque reduction The percentage reduction in viral growth at varying concentrations of the test compounds is depicted in (Fig. 8 b). The results are presented as percentage reduction in plaque count relative to the drug concentration. Antiviral activity was evaluated for non-cytotoxic concentrations (2 µM and 160 µM) of the test compounds. At 2 µM, none of the compounds exhibited significant antiviral effect, except ACY-NPs which showed 23% inhibition. Further, ACY- NPs demonstrated a notable reduction in plaques, starting from 5 µM, with complete inhibition (100%) at 20 µM. This was better than the standard, ACY, which showed 100% inhibition at 160 µM. In contrast, ACPD and ACPD-NPs exhibited minimal antiviral activity compared to both ACY and ACY-NPs, achieving only 50% plaque reduction at the highest concentration (160 µM) tested. These findings were further validated by esterase enzyme activity assays, which were conducted to analyse the observed differences. ACPD to ACY through enzymatic cleavage At the beginning of the experiment (time 0 minutes), ACPD concentration was at its maximum. Over time, esterase activity gradually cleaved the ester bond in ACPD, yielding the active drug ACY. The retention times for ACY and ACPD were 2.76 minutes and 8.49 minutes, respectively, and by 90 minutes, most of ACPD had been cleaved into ACY, as shown in Supplementary Fig. S15. In the plaque reduction assay, ACPD showed only 50% inhibition at 160 µM, which was attributed to delayed cleavage due to the absence of esterase enzyme in Vero cells and in the culture medium. Although ester bonds can undergo hydrolytic cleavage, the process is very slow. To establish the proof of concept of conversion of ACPD to ACY through esterase enzyme, in vitro experiment using porcine liver esterase was performed, which successfully cleaved ACPD into ACY and verified by RP-HPLC. These findings highlight the critical role of corneal esterase in converting any esterified prodrug to active drug is reported previously [ 17 , 42 ]. From our findings, it is suggested that presence of esterase enzymes in in-vivo can facilitate ACPD cleavage, potentially enhance the therapeutic efficacy of ACPD. Esterase-mediated cleavage of ester bonds is critical for the breakdown of prodrug to drug and controlled release of active pharmaceutical ingredients in ocular drug delivery systems[ 18 ]. Understanding esterase activity in ocular tissues is essential for designing prodrugs and polymeric carriers that depend on enzymatic activation for therapeutic efficacy. This mechanism enables site-specific drug release and reduces systemic associated side effects. For example, latanoprost, a prostaglandin analogue, relies on corneal epithelial esterase to cleave its ester bonds and release the active compound, as demonstrated in reported studies [ 43 ]. CONCLUSION In the present study, the ACY NPs were formed by applying a simple and efficient nanoprecipitation method. ACPD was synthesized and encapsulated successfully in PLGA nanoparticles using single emulsion solvent evaporation method. The formulated ACY and ACPD-loaded PLGA NPs exhibited almost uniform particle size, smooth surface morphology, and good surface charge. The formulated NPs are found to be appropriate for treating HSK. ACY NPs demonstrated significantly higher efficacy in inhibiting HSV growth compared to free ACY in Vero cells. ACPD-NPs, showed a more stable and controlled release profile across various pH conditions. Esterase enzyme assay confirmed the cleavage of ACPD to ACY and these findings suggest that the presence of esterase enzymes in animals can facilitate the cleavage of ACPD, potentially enhancing the therapeutic efficacy of ACPD. This nanoparticle formulation presents an efficient delivery system for the treatment of ocular viral infections. Future in vivo studies will further confirm the effectiveness of this nanocarrier-based drug delivery system to treat HSK. Declarations Author contributions S.S.H: Data curation, Investigation, Formal analysis, Writing – original draft & editing. D.V. P: Data curation, Investigation, Formal analysis, Writing – original draft & editing. S.L.G: Data curation, writing and editing. K.K: Data curation, writing and editing. P.P.M: Data curation, writing and editing. Y.N.N: Data curation, writing and editing. B.R.G : Conceptualization, Supervision, writing – review, editing and final approval of the manuscript. All the authors read the final manuscript and approved for the publication. Acknowledgements We thank the Manipal Center for Infectious Diseases (MAC ID) for awarding intramural grant for this research work. We thank Manipal Institute of Technology, Manipal and Manipal Institute of Virology, Manipal for allowing to conduct this research. We thank Manipal Academy of Higher Education, Manipal for proving Dr. TMA Pai scholarship to D.V.P. Competing interests No potential conflict of interest was reported by the author(s). Data availability statement Data is presented in the main manuscript and supplementary information. Funding: Manipal Center for Infectious Diseases (MAC ID), an intramural grant from Manipal Academy of Higher Education (MAHE), Manipal. References Herpes simplex virus n.d. accessed April 27, (2025). https://www.who.int/news-room/fact-sheets/detail/herpes-simplex-virus# McCormick, I. et al. Ophthalmic Epidemiol. ; 29 :353. https://doi.org/10.1080/09286586.2021.1962919 . (2021). Maroui, M. A. et al. Latency Entry of Herpes Simplex Virus 1 Is Determined by the Interaction of Its Genome with the Nuclear Environment. PLoS Pathog . 12 , e1005834. https://doi.org/10.1371/JOURNAL.PPAT.1005834 (2016). Al-Dujaili, L. J. et al. Ocular herpes simplex virus: how are latency, reactivation, recurrent disease and therapy interrelated? Future Microbiol. 6 , 877. https://doi.org/10.2217/FMB.11.73 (2011). Kimberlin, D. W. & Whitley, R. J. Antiviral therapy of HSV-1 and – 2. Hum. Herpesviruses: Biology Therapy Immunoprophylaxis 2007 :1153–1174. https://doi.org/10.1017/CBO9780511545313.065 Poole, C. L. & James, S. H. Antiviral Therapies for Herpesviruses: Current Agents and New Directions. Clin. Ther. 40 , 1282. https://doi.org/10.1016/J.CLINTHERA.2018.07.006 (2018). LeBlanc, R. A., Pesnicak, L., Godleski, M. & Straus, S. E. The Comparative Effects of Famciclovir and Valacyclovir on Herpes Simplex Virus Type 1 Infection, Latency, and Reactivation in Mice. J. Infect. Dis. 180 , 594–599. https://doi.org/10.1086/314962 (1999). Hawthorne, K. M., Dana, R. & Chodosh, J. Delayed Type Hypersensitivity in the Pathogenesis of Recurrent Herpes Stromal Keratitis. Semin Ophthalmol. 26 , 246–250. https://doi.org/10.3109/08820538.2011.588659 (2011). Stanfield, B. & Kousoulas, K. G. Herpes Simplex Vaccines: Prospects of Live-attenuated HSV Vaccines to Combat Genital and Ocular infections. Curr. Clin. Microbiol. Rep. 2 , 125. https://doi.org/10.1007/S40588-015-0020-4 (2015). Van Wagoner, N., Qushair, F. & Johnston, C. Genital Herpes Infection: Progress and Problems. Infect. Dis. Clin. North. Am. 37 , 351–367. https://doi.org/10.1016/J.IDC.2023.02.011 (2023). Alvaro, P. M., Sitar, D. S. & Aoki, F. Y. Comparative bioavailability of acyclovir from oral valacyclovir and acyclovir in patients treated for recurrent genital herpes simplex virus infection. Can. J. Clin. Pharmacol. 8 , 207–211 (2001). Donalisio, M. et al. Acyclovir-Loaded Chitosan Nanospheres from Nano-Emulsion Templating for the Topical Treatment of Herpesviruses Infections. Pharmaceutics 10 , 46. https://doi.org/10.3390/PHARMACEUTICS10020046 (2018). Frejborg, F., Kalke, K. & Hukkanen, V. Current landscape in antiviral drug development against herpes simplex virus infections 2022. https://doi.org/10.1002/SMMD.20220004 Yadav, K. S. et al. Microemulsions for enhancing drug delivery of hydrophilic drugs: Exploring various routes of administration. Med. Drug Discov . 20 , 100162. https://doi.org/10.1016/J.MEDIDD.2023.100162 (2023). Freeman, D. J., Sheth, N. V. & Spruance, S. L. Failure of topical acyclovir in ointment to penetrate human skin. Antimicrob. Agents Chemother. 29 , 730–732. https://doi.org/10.1128/AAC.29.5.730 (1986). Soul-Lawton, J. et al. Absolute bioavailability and metabolic disposition of valaciclovir, the L-valyl ester of acyclovir, following oral administration to humans. Antimicrob. Agents Chemother. 39 , 2759–2764. https://doi.org/10.1128/AAC.39.12.2759 (1995). Heikkinen, E. M. et al. Esterase activity in porcine and albino rabbit ocular tissues. Eur. J. Pharm. Sci. 123 , 106–110. https://doi.org/10.1016/J.EJPS.2018.07.034 (2018). Hammid, A. et al. Activity and Expression of Carboxylesterases and Arylacetamide Deacetylase in Human Ocular Tissues. Drug Metab. Dispos. 50 , 1483–1492. https://doi.org/10.1124/DMD.122.000993 (2022). Perez, C., Daniel, K. B. & Cohen, S. M. Evaluating Prodrug Strategies for Esterase-Triggered Release of Alcohols. ChemMedChem. ;8:1662. (2013). https://doi.org/10.1002/CMDC.201300255 Mahboobian, M. M., Mohammadi, M. & Mansouri, Z. Development of thermosensitive in situ gel nanoemulsions for ocular delivery of acyclovir. J. Drug Deliv Sci. Technol. 55. https://doi.org/10.1016/J.JDDST.2019.101400 (2020). Donalisio, M. et al. Acyclovir-Loaded Chitosan Nanospheres from Nano-Emulsion Templating for the Topical Treatment of Herpesviruses Infections. Pharmaceutics 10 , 46. https://doi.org/10.3390/PHARMACEUTICS10020046 (2018). Merkli, A., Tabatabay, C., Gurny, R. & Heller, J. Biodegradable polymers for the controlled release of ocular drugs. Prog Polym. Sci. 23 , 563–580. https://doi.org/10.1016/S0079-6700(97)00048-8 (1998). Huang, Y. et al. Nanotechnology’s frontier in combatting infectious and inflammatory diseases: prevention and treatment. Signal. Transduct. Target. Therapy 2024 . 9 , 1. https://doi.org/10.1038/s41392-024-01745-z (2024). Blecher, K., Nasir, A. & Friedman, A. The growing role of nanotechnology in combating infectious disease. Virulence 2 , 395–401. https://doi.org/10.4161/VIRU.2.5.17035 (2011). Hakkimane, S. S., Shenoy, V. P., Gaonkar, S. L., Bairy, I. & Guru, B. R. Antimycobacterial susceptibility evaluation of rifampicin and isoniazid benz-hydrazone in biodegradable polymeric nanoparticles against Mycobacterium tuberculosis H37Rv strain. Int. J. Nanomed. 13 , 4303–4318. https://doi.org/10.2147/IJN.S163925 (2018). Praveena, J. & Guru, B. R. Simultaneous estimation of paclitaxel and curcumin in nano-formulation: Stability analysis of drugs, optimization and validation of HPLC method. J. Appl. Pharm. Sci. 11 ,, 071–83. https://doi.org/10.7324/JAPS.2021.110308 (2021). Hakkimane, S. S. & Guru, B. R. Nano formulation analysis: Analytical method development of isoniazid and simultaneous estimation of antitubercular drugs isoniazid and rifampicin by reverse phase high pressure liquid chromatography. Asian J. Pharm. Clin. Res. 10 , 330–335. https://doi.org/10.22159/AJPCR.2017.V10I5.17582 (2017). Govender, T., Stolnik, S., Garnett, M. C., Illum, L. & Davis, S. S. PLGA nanoparticles prepared by nanoprecipitation: drug loading and release studies of a water soluble drug. J. Control Release . 57 , 171–185. https://doi.org/10.1016/S0168-3659(98)00116-3 (1999). Muszalska-Kolos, I., Lesniewska-Kowiel, M. A., Plewa, S. & Klupczyńska, A. Tricyclic Derivative of Acyclovir and Its Esters in Relation to the Esters of Acyclovir Enzymatic Stability: Enzymatic Stability Study. Molecules 2020;25. https://doi.org/10.3390/MOLECULES25092156 INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE ICH HARMONISED TRIPARTITE GUIDELINE VALIDATION OF ANALYTICAL PROCEDURES. TEXT AND METHODOLOGY Q2(R1) n.d. Aboelezz, A., Kharouba, M. & Mahmoud, S. H. A simple method for the determination of acyclovir concentrations in human plasma using high-performance liquid chromatography. Future J. Pharm. Sci. 2024 . 10 , 1. https://doi.org/10.1186/S43094-024-00649-7 (2024). Praveena, J., Hakkimane, S. S. & Guru, B. R. Synergistic effect of Paclitaxel and Curcumin in nano-formulations on U87 and A549 cancer cell lines. J. Appl. Pharm. Sci. 12 ,, 031–47. https://doi.org/10.7324/JAPS.2021.120204 (2022). Hakkimane, S. S., Shenoy, V. P., Gaonkar, S. L., Bairy, I. & Guru, B. R. Antimycobacterial susceptibility evaluation of rifampicin and isoniazid benz-hydrazone in biodegradable polymeric nanoparticles against Mycobacterium tuberculosis H37Rv strain. Int. J. Nanomed. 13 , 4303–4318. https://doi.org/10.2147/IJN.S163925 (2018). Pinto, J., Ahmad, M. & Guru, B. R. Enhancing the efficacy of fluocinolone acetonide by encapsulating with PLGA nanoparticles and conjugating with linear PEG polymer. J. Biomater. Sci. Polym. Ed. 30 , 1188–1211. https://doi.org/10.1080/09205063.2019.1625524 (2019). Segets, D. et al. Experimental and theoretical studies of the colloidal stability of nanoparticles-a general interpretation based on stability maps. ACS Nano . 5 , 4658–4669. https://doi.org/10.1021/NN200465B (2011). Sinha, V. R., Monika, Trehan, A., Kumar, M., Singh, S. & Bhinge, J. R. Stress studies on acyclovir. J. Chromatogr. Sci. 45 , 319–324. https://doi.org/10.1093/CHROMSCI/45.6.319 (2007). Jain, A. et al. Peptide and protein delivery using new drug delivery systems. Crit. Rev. Ther. Drug Carrier Syst. 30 , 293–329. https://doi.org/10.1615/CRITREVTHERDRUGCARRIERSYST.2013006955 (2013). Makadia, H. K. & Siegel, S. J. Poly Lactic-co-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier. Polym. (Basel) . 3 , 1377. https://doi.org/10.3390/POLYM3031377 (2011). Alsaab, H. O. et al. PLGA-Based Nanomedicine: History of Advancement and Development in Clinical Applications of Multiple Diseases. Pharmaceutics 14 , 2728. https://doi.org/10.3390/PHARMACEUTICS14122728 (2022). Wang, F. et al. The Hydrolysis of Diclofenac Esters: Synthetic Prodrug Building Blocks for Biodegradable Drug-Polymer Conjugates. J. Pharm. Sci. 105 , 773–785. https://doi.org/10.1002/jps.24665 (2016). Kuny, C. V., Bowen, C. D., Renner, D. W., Johnston, C. M. & Szpara, M. L. In vitro evolution of herpes simplex virus 1 (HSV-1) reveals selection for syncytia and other minor variants in cell culture. Virus Evol. 6. https://doi.org/10.1093/VE/VEAA013 (2020). Akkurt Arslan, M. et al. Profiling tear film enzymes reveals major metabolic pathways involved in the homeostasis of the ocular surface. Sci. Rep. 2023 . 13 , 1. https://doi.org/10.1038/s41598-023-42104-2 (2023). Tripathy, K., Patel, P., Geetha, R., Latanoprost & XPharm Compr. Pharmacol. Ref. :1–4. https://doi.org/10.1016/B978-008055232-3.62015-X . (2024). Additional Declarations No competing interests reported. Supplementary Files Supplementaryinformationsubmission.pdf Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 13 Apr, 2026 Reviews received at journal 09 Apr, 2026 Reviewers agreed at journal 31 Mar, 2026 Reviews received at journal 23 Jan, 2026 Reviewers agreed at journal 14 Jan, 2026 Reviews received at journal 19 Sep, 2025 Reviewers agreed at journal 15 Sep, 2025 Reviewers invited by journal 10 Sep, 2025 Editor assigned by journal 10 Sep, 2025 Editor invited by journal 03 Sep, 2025 Submission checks completed at journal 30 Aug, 2025 First submitted to journal 30 Aug, 2025 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7427697","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":515262288,"identity":"b0494773-4304-46d1-b324-d42fdc104706","order_by":0,"name":"Sushruta S Hakkimane","email":"","orcid":"","institution":"Manipal Institute of Technology Bengaluru, Manipal Academy of Higher Education Manipal","correspondingAuthor":false,"prefix":"","firstName":"Sushruta","middleName":"S","lastName":"Hakkimane","suffix":""},{"id":515262289,"identity":"a7d1d2b3-8e98-451b-bd1a-80fb47ff05b9","order_by":1,"name":"Divakarareddy Vemanna Paladugulu","email":"","orcid":"","institution":"Manipal Institute of Technology, Manipal Academy of Higher Education Manipal","correspondingAuthor":false,"prefix":"","firstName":"Divakarareddy","middleName":"Vemanna","lastName":"Paladugulu","suffix":""},{"id":515262290,"identity":"8805032c-90c8-4a75-916b-0a1612971d6b","order_by":2,"name":"Santosh L Gaonkar","email":"","orcid":"","institution":"Manipal Institute of Technology, Manipal Academy of Higher Education Manipal","correspondingAuthor":false,"prefix":"","firstName":"Santosh","middleName":"L","lastName":"Gaonkar","suffix":""},{"id":515262291,"identity":"cc737049-6702-44e2-8bac-b7e78b018c43","order_by":3,"name":"Kavitha Karunakaran","email":"","orcid":"","institution":"Manipal Institute of Virology, Manipal Academy of Higher Education Manipal","correspondingAuthor":false,"prefix":"","firstName":"Kavitha","middleName":"","lastName":"Karunakaran","suffix":""},{"id":515262292,"identity":"3dd0763b-6462-4dc5-a8b1-8bde99adf6cc","order_by":4,"name":"Piya Paul Mudgal","email":"","orcid":"","institution":"Manipal Institute of Virology, Manipal Academy of Higher Education Manipal","correspondingAuthor":false,"prefix":"","firstName":"Piya","middleName":"Paul","lastName":"Mudgal","suffix":""},{"id":515262293,"identity":"2430ba1b-f416-4629-959d-a25fb085fc8f","order_by":5,"name":"Yogeesha N Nayak","email":"","orcid":"","institution":"Manipal Institute of Technology, Manipal Academy of Higher Education Manipal","correspondingAuthor":false,"prefix":"","firstName":"Yogeesha","middleName":"N","lastName":"Nayak","suffix":""},{"id":515262297,"identity":"cc7d5c0d-f3cb-454b-a663-688da129e4c0","order_by":6,"name":"Bharath Raja Guru","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEklEQVRIiWNgGAWjYBACewkwdQDC+wDEbAwMjEAuM04thjNAZAJEC+MMiBYGvFoMbiBpYeaBiuK3ZXbzsw8/f9xhkI/IfSZtm2OXxyfd/OAAQ4V1YgMuv8gcM57Zk/CMwfBGupl07rbkYjaZYwYHGM6k49RiOCPBmIEn4TCQkcYG1MKc2CaRYHCAse0wTi0GN9I/M/6BabHcVg/Ukv7hAOM/fFpyjJlBtshLALUwbjsM1JIDtKUBtxbDGTnFzDJph3kMeJ4xW/ZuO57YJnOm4EDCsXRjnN6XSN/M+MbmsJx8exrjjZ/bqhPnz27f+OBDjbUsLi0wwAMMJSgApYcEAsrBQB5uqAQxykfBKBgFo2AkAQC0mVzcbdReawAAAABJRU5ErkJggg==","orcid":"","institution":"Manipal Institute of Technology, Manipal Academy of Higher Education Manipal","correspondingAuthor":true,"prefix":"","firstName":"Bharath","middleName":"Raja","lastName":"Guru","suffix":""}],"badges":[],"createdAt":"2025-08-21 15:38:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7427697/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7427697/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91556177,"identity":"8f707914-c42b-433f-ada3-743ae648a971","added_by":"auto","created_at":"2025-09-17 17:24:39","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":24552,"visible":true,"origin":"","legend":"\u003cp\u003eAcyclovir Structure\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7427697/v1/6b124c54e4249fc7bb8bb0a4.jpeg"},{"id":91556413,"identity":"31852724-0f35-43b6-83b7-dd49b09fd484","added_by":"auto","created_at":"2025-09-17 17:32:39","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":36684,"visible":true,"origin":"","legend":"\u003cp\u003eSynthesis of 2-((2-amino-6-oxo-3,6-dihydro-9\u003cem\u003eH\u003c/em\u003e-purin-9-yl)methoxy)ethyl benzoate \u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7427697/v1/636b3bed71b6c35a8c2281ee.jpeg"},{"id":91556414,"identity":"dc1c584e-ac37-4048-8216-688c0ed5d9c6","added_by":"auto","created_at":"2025-09-17 17:32:39","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":139067,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eFTIR spectrum of ACY and ACPD (3). \u003cstrong\u003e(b) \u003c/strong\u003eExpanded \u003csup\u003e1\u003c/sup\u003eH-NMR of OH, NH \u0026amp; NH\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e \u003c/sup\u003eof ACY and ACPD which indicates the disappearance of OH peak in the product while the NH \u0026amp; NH\u003csub\u003e2\u003c/sub\u003e remains intact.\u003cstrong\u003e (c) \u003c/strong\u003eThe \u003csup\u003e13\u003c/sup\u003eC NMR spectrum of ACPD\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7427697/v1/0726affc01efc740ed6e1985.jpeg"},{"id":91556178,"identity":"8efa8a46-e94c-4a67-b3b9-07d0d5b8f5f3","added_by":"auto","created_at":"2025-09-17 17:24:39","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":74934,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eChromatogram of ACY with a retention time of 3.865 min. \u003cstrong\u003e(b) \u003c/strong\u003eChromatogram of ACPD at RT 8.477 min. \u003cstrong\u003e(c) \u003c/strong\u003eSimultaneous estimation of ACY (RT-2.767) and ACPD (RT-8.948).\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7427697/v1/8e73a6d0e6d92ddc1328dee8.jpeg"},{"id":91556187,"identity":"f322815e-a23c-49a4-a12c-32ec22e4cb8d","added_by":"auto","created_at":"2025-09-17 17:24:39","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":414287,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eParticle size distribution graph of prepared ACY-PLGA NPs.\u003cstrong\u003e (b) \u003c/strong\u003eZeta graph of prepared ACY-PLGA NPs showing negative surface charge. \u003cstrong\u003e(c) \u003c/strong\u003eParticle size distribution graph of prepared ACPD-PLGA NPs. \u003cstrong\u003e(d) \u003c/strong\u003eZeta graph of prepared ACPD-PLGA NPs showing negative surface charge. \u003cstrong\u003e(e) \u003c/strong\u003eSEM image of ACPD-loaded NPs showing a smooth surface and size\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7427697/v1/f9a16587696b94bb1a469339.jpeg"},{"id":91556961,"identity":"270b0f2d-e7b7-4fdd-8ddd-386610c6c0f3","added_by":"auto","created_at":"2025-09-17 17:40:39","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":138866,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eStability of ACY at pH 7.4, 4.5 and 5.2. \u003cstrong\u003e(b) \u003c/strong\u003eStability of ACPD at pH 7.4, 4.5 and 5.2\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7427697/v1/3f61115e52b1f04133321271.jpeg"},{"id":91556415,"identity":"39111405-9626-420d-8d82-5e6634601d48","added_by":"auto","created_at":"2025-09-17 17:32:39","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":112658,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eCumulative \u003cem\u003ein vitro\u003c/em\u003erelease of ACY from PLGA NPs. \u003cstrong\u003e(b)\u003c/strong\u003e Cumulative \u003cem\u003ein vitro\u003c/em\u003e release of ACPD from PLGA NPs\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7427697/v1/e2f3499805b6509a522933fa.jpeg"},{"id":91556186,"identity":"8cb5dbf6-c215-4be7-9748-f89574721640","added_by":"auto","created_at":"2025-09-17 17:24:39","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":243882,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eCytotoxicity profile of varying concentrations of test formulations. \u003cstrong\u003e(b)\u003c/strong\u003e Percentage reduction in plaques by test formulations\u003c/p\u003e","description":"","filename":"image8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7427697/v1/ee807fae39271b14125cd32d.jpeg"},{"id":91557784,"identity":"de6219da-e7b6-4baa-acb2-9d0b67c2bed8","added_by":"auto","created_at":"2025-09-17 17:56:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2358626,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7427697/v1/94225298-6df2-4941-a156-50d5a9e075d2.pdf"},{"id":91557318,"identity":"b12130af-728e-433b-8b0e-a528bef07b79","added_by":"auto","created_at":"2025-09-17 17:48:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":6588695,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformationsubmission.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7427697/v1/54d35b42ad466ef5f37ad3c8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biodegradable Polymeric Nano formulation of Acyclovir and Acyclovir prodrug for Enhanced Therapeutic Efficacy Against Herpes Simplex Virus Infections","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eHerpes simplex virus (HSV) is a common viral infection that mainly causes oral herpes, genital herpes and ocular infections like keratitis. HSV infections are widespread, posing a significant clinical challenge worldwide. HSV-1 and HSV-2 infections affect over two-thirds of the global population, with rising incidences. As per the WHO estimates in 2020, HSV-1 had infected 3.8\u0026nbsp;billion people under the age of 50[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Around 1.8\u0026nbsp;million people were affected with herpes simplex keratitis (HSK), caused by HSV-1 and leads to corneal inflammation, scarring and vision impairment[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Herpes simplex keratitis (HSK) is one of the leading causes of infectious blindness. As HSV-1 undergoes the latency stage after a primary infection, it enters and hides in sensory ganglia[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Certain factors like surgery, stress, and immunosuppression can reactivate the virus, causing the recurrence of infection, which leads to vision impairment and vision loss[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. To inhibit HSV-1, antiviral drugs like acyclovir, valacyclovir, and famciclovir are used. These are delivered orally or topically and often face challenges like poor drug delivery due to barriers, latency, poor bioavailability, premature degradation, short half-lives, and low penetrating capacity to certain cellular sites[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These challenges pose a threat of recurrence and cause corneal damage in HSK patients[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The oral treatment may extend up to 6 to 9 months, leading to various side effects and drug resistance. Despite ongoing efforts, no approved vaccines are available yet, though several candidates show evident results in early clinical trials [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Several barriers and several factors hamper the rapid advancement of antiviral drugs. There is a constant need for the development of novel medications and the enhancement of existing drug formulations. Among the currently available antiviral agents, acyclovir (ACY) remains the most widely used treatment for HSV-related infections.\u003c/p\u003e\u003cp\u003eAcyclovir (ACY) is one of the first-line treatments for HSV-1. ACY is a synthetic purine nucleoside analogue with the chemical formula C₈H₁₁N₅O₃ and a molecular weight of 225.20 g/mol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The Food Drug Administration (FDA) has approved it for treating genital herpes and HSV encephalitis due to its affordability, safety, and tolerability[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. ACY requires intracellular activation to exert its antiviral effects. Viral thymidine kinase (TK, UL23 gene) catalyses the conversion of ACY into ACY monophosphate, which is further phosphorylated to form ACY triphosphate. This process increases ACY concentration in infected cells while reducing cellular efflux, thereby enhancing its antiviral activity[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. ACY effectively inhibits viral DNA replication and synthesis. Although it reduces symptoms and recurrences, ACY does not eliminate the virus[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThough there are many topical ACY formulations, they fail to reach the infected site due to various physiological factors. As ACY is hydrophilic, it cannot pass through the lipophilic barriers to reach HSV-1[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Even the topical creams and gels have poor retention, leading to short exposure time[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. ACY has low bioavailability; only a small fraction will reach infected cells. ACY will fail to reach the latent virus as it does not penetrate through the deeper layer of tissue and neurons. Due to a shorter half-life, ACY is rapidly eliminated, so frequent dosing is needed. To overcome ACY\u0026rsquo;s limitations, the derivatives, such as valacyclovir (VACV), the L-valyl ester of ACY, have been developed to improve bioavailability following oral administration[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Even though they require fewer doses compared to acyclovir, the higher systemic levels will cause renal toxicity and other side effects, like headache, and they also require a long-term treatment. To overcome these limitations, we are aiming to develop esterified ACPD, which serves as proof of concept to treat herpes simplex keratitis (HSK) caused by HSV-1. In the cornea of the eye, there are esterase enzymes that help to cleave ACPD to ACY[\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and ACPD will be converted to an active form only at the site of the action. This will increase the stability and the maximum amount of drug will reach the infected site. Even though there are wide treatment strategies to treat herpetic eye diseases, no intracameral formulation is available for the treatment of stromal and endothelial infected herpetic eye disease. Therefore, the intracameral formulations like anterior Chamber (AC) injection with ACY or ACPD NPs or implant with sustained release directly at the local tissue can reduce systemic side effects and increases the effectiveness of treating HSV-1[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNanotechnology-driven formulations present a novel approach for improving the localised efficacy of antiviral agents and advancing therapeutic strategies for viral infections. These formulations improve drug solubility, protect the drug from degradation, and help overcome biological barriers. Additionally, they enable controlled and sustained drug release at the site of infection, enhancing therapeutic outcomes[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. As reported in the literature, numerous attempts have been made to alter the pharmacokinetics and improve bioavailability of antiviral medications by making it as prodrugs. Prodrugs also improve the physicochemical characteristics like solubility, bioavailability and stability, which creates more efficient approach of treating viral diseases by overcoming the limitations of traditional antiviral medications[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. A combination of prodrugs and nanotechnology will be a better strategy to treat HSV-1. Nano-formulations are a potential strategy to enhance the therapeutic efficacy by creating controlled drug delivery using nanoparticulate carriers. Our previous work from the lab supports prodrugs are more stable in different pH conditions and they are as effective as pure drug at the same molar concentration. Drug and prodrug encapsulated in NPs are more effective in suppressing the growth of tuberculosis strain at much lower concentrations compared to pure drug [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The findings encouraged us to adapt the similar strategy to improve the effective treatment for the herpes virus infections. The combination of nano-formulations with prodrug will yield better efficacy compared to individual treatment strategies.\u003c/p\u003e\u003cp\u003eIn this study, prodrug of ACY is prepared by reacting benzoic acid with ACY which creates hydrolytically cleavable ester bond with ACY, referred to as ACPD in this manuscript. ACPD-loaded PLGA NPs were prepared, which increases the stability and its encapsulation in NPs. Further, assessed their cytotoxicity and plaque reduction activity compared to pure ACY and ACY NPs. We also conducted ACPD in \u003cem\u003evitro\u003c/em\u003e studies with calculated amount of esterase enzyme in buffer. the results show cleavage of ACPD to ACY and it is measured and quantified using RP-HPLC.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003eAll reagents were obtained from commercial suppliers and used without further purification. The infrared spectrum of ACPD was recorded using a Shimadzu FTIR spectrophotometer. The \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz) and \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz) spectra were acquired on a Bruker AM 400 MHz NMR spectrometer with TMS as an internal standard. High-resolution mass spectrometry (HRMS) analysis was performed via the time of flight (TOF) and electrospray ionization (ESI) method. Reaction progress and completion were monitored by thin layer chromatography (TLC) on silica gel plates, with spot visualization under UV light (365 nm). The melting points of the compounds were determined using the open capillary method.\u003c/p\u003e\u003cp\u003ePoly(D, L-lactide-co-glycolide) (PLGA 50:50; molecular weight: 100,000\u0026ndash;120,000 Da) was purchased from Durect Corporation (Birmingham, AL, USA). Acetonitrile and methanol were obtained from Merck Millipore, India. Acyclovir (ACY) was kindly gifted by Zydus Lifesciences Ltd. (Ahmedabad, India). Dimethyl sulfoxide (DMSO), 4-dimethylaminopyridine (DMAP), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were purchased from Spectrochem Pvt. Ltd. (Mumbai, India). Benzoic acid and TLC plates were obtained from Merck Millipore, India. All other reagents and solvents used were of HPLC grade. Polyvinyl alcohol (PVA, 87\u0026ndash;89% hydrolyzed; molecular weight: ~120,000 Da) was purchased from Thomas Baker (Mumbai, India). Cell culture reagents included Minimal Essential Medium (MEM), 96-well plates, and 12-well plates from HiMedia (Thane, India), and fetal bovine serum (FBS) from Gibco (Mumbai, India). Trypsin, sterile phosphate-buffered saline (PBS), and Trypan Blue were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Vero cells (ATCC\u0026reg; CCL-81\u0026trade;), procured from the American Type Culture Collection (ATCC) and the HSV-1 MacIntyre strain (ATCC\u0026reg; VR-539\u0026trade;) were used in antiviral studies. Agarose overlay medium and esterase from porcine liver (molecular weight: 168 kDa) were procured from Sigma-Aldrich, Merck (Darmstadt, Germany).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eChemistry\u003c/h2\u003e\u003cp\u003e\u003cb\u003eSynthesis of 2-((2-amino-6-oxo-3,6-dihydro-9\u003c/b\u003e\u003cb\u003eH\u003c/b\u003e\u003cb\u003e-purin-9-yl)methoxy)ethyl benzoate (ACPD)(3)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAn oven-dried, 50 mL round-bottom flask equipped with a magnetic stir bar was charged with ACY (0.44 mmol), benzoic acid (0.44 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 0.55 mmol), and 4-dimethylaminopyridine (DMAP, 0.46 mmol), serving as a coupling reagent and catalyst, respectively. To this, 2 ml of anhydrous DMF was added under nitrogen environment to maintain the anhydrous conditions. The reaction mixture was stirred at room temperature overnight. The reaction progress was monitored by TLC using a methanol: chloroform (1:9) solvent system. Upon completion, the reaction mixture was quenched with cold water, resulting in the formation of a precipitate. The precipitate was filtered, dried, and subsequently recrystallised from isopropanol.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSpectral Data:\u003c/h3\u003e\n\u003cp\u003e\u003cb\u003e2-((2-amino-6-oxo-3,6-dihydro-9\u003c/b\u003e\u003cb\u003eH\u003c/b\u003e\u003cb\u003e-purin-9-yl)methoxy)ethyl benzoate (3)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWhite solid, yield\u0026thinsp;=\u0026thinsp;88%, MP\u0026thinsp;=\u0026thinsp;227\u0026ndash;229\u0026deg;C, FTIR (cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e): 3447 \u0026amp; 3319 (NH\u003csub\u003e2\u003c/sub\u003e stretching), 3194 (NH stretching), 3101 (CH stretching aromatic), 2928 (CH stretching aliphatic), 1713 (C\u0026thinsp;=\u0026thinsp;O ester), 1687 (C\u0026thinsp;=\u0026thinsp;O pyrimidine),1604 (N\u0026thinsp;=\u0026thinsp;C stretching), 1572 (C\u0026thinsp;=\u0026thinsp;C stretching), 1386 (CH\u003csub\u003e2\u003c/sub\u003e bending), 1277 (C-O-C stretching). \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO) δ 10.63 (s, 1H), 7.87 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 3H), 7.66 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 1H), 7.52 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7 Hz, 2H), 6.51 (s, 2H), 5.41 (s, 2H), 4.36 (s, 2H), 3.82 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.2 Hz, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO) δ 166.08, 157.27, 154.37, 151.91, 138.21, 133.84, 129.94, 129.57, 129.20, 117.05, 72.35, 66.92, 64.02. HRMS (TOF): 330.16 (M\u0026thinsp;+\u0026thinsp;H)\u003csup\u003e+\u003c/sup\u003e, 330.12 (Calculated M\u0026thinsp;+\u0026thinsp;H); MF: C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e.\u003c/p\u003e\n\u003ch3\u003eQuantitative Analysis by Liquid Chromatography\u003c/h3\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eOptimisation and Method Development for ACY and ACPD\u003c/h2\u003e\u003cp\u003eThe analysis was performed on a reverse-phase high-performance liquid chromatography (RP-HPLC) system (Shimadzu LC-20AD), integrated with an autosampler (SIL-20AC), a PDA detector (SPD-M40). Chromatographic separation was performed using a Phenomenex\u0026reg; Luna C18 column (5 \u0026micro;m particle size, 100 \u0026Aring; pore diameter, 250 mm length), followed by data acquisition and processing using LabSolutions software (Shimadzu, Kyoto, Japan). Binary gradient mode with an injection volume of 20 \u0026micro;L was used. All solvents and reagents employed were of HPLC grade to ensure analytical precision. For ACY analysis, the mobile phase was a mixture of 10:90 acetonitrile and water, with a flow rate of 1 mL/min, and a run time of 10 minutes, with detection performed at 254 nm. A stock solution (1 mg/mL) was prepared by dissolving 10 mg of the drug in 10 mL of water, and a calibration curve was constructed using different concentrations.\u003c/p\u003e\u003cp\u003eACPD was analysed using a reverse-phase HPLC method with a binary gradient mobile phase of acetonitrile and water. The flow rate was maintained at 1.0 mL/min, and the total run time was 16 minutes. The gradient flow started with 20% acetonitrile for 1 minute, increased to 70% by 5 minutes, then reduced back to 20% by 8 minutes, and held at 20% until 16 minutes for system stabilisation. Detection was carried out at 245 nm. A stock solution of ACPD (1 mg/mL) was prepared by dissolving 10 mg of the compound in 10 mL of acetonitrile containing 1% DMSO. A calibration curve was constructed using serial dilutions of this stock. The same method was used for the simultaneous estimation of ACPD and ACY.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMethod validation\u003c/h3\u003e\n\u003cp\u003eFollowing system equilibration, three blank injections were performed to ensure baseline stability. System suitability was confirmed by injecting five replicates of a calibration standard solution of ACY. Method development and validation were conducted as per established procedures [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The analysis was performed with an injection volume of 20 \u0026micro;L, a total run time of 10 minutes, and detection at 254 nm. This method was successfully used to analyse ACY in nanoparticle drug formulations. Validation parameters such as linearity, precision, and repeatability were evaluated as per ICH guideline Q2(R1) (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use). Repeatability was checked using intraday variation, while intermediate precision was assessed through inter-day variation. Overall precision included repeatability, intermediate precision, and reproducibility.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePreparation of ACPD and ACY loaded nanoparticles\u003c/h2\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003eSingle Emulsion solvent evaporation Technique to make ACPD NPs\u003c/h2\u003e\u003cp\u003eACPD-loaded PLGA nanoparticles were prepared by the single emulsion solvent evaporation method. PLGA and ACPD (2:1, w/w) were dissolved in chloroform to form the organic phase, which was stirred (SCHOTT Instruments, Mainz, Germany) and added dropwise into 12 mL of 2.5% (w/v) PVA solution kept in an ice bath. The mixture was sonicated using a probe sonicator (QSonica CL-334, Newtown, CT, USA) at 40% amplitude for 6 minutes to form an oil-in-water emulsion. The emulsion was stirred for 12 hours at room temperature to evaporate the organic solvent. Nanoparticles were collected by centrifugation (Eppendorf 5430R, Hamburg, Germany) at 11,000 rpm for 30 minutes at 4\u0026deg;C. The pellet was washed, redispersed in deionised water, and lyophilised (LaboGene ScanVac CoolSafe, Lynge, Denmark) at \u0026minus;\u0026thinsp;108\u0026deg;C. The dried nanoparticles were stored at \u0026minus;\u0026thinsp;20\u0026deg;C. Blank nanoparticles (without drug) were prepared using the same procedure, with only PLGA in chloroform [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003eNanoprecipitation technique to make ACY NPs\u003c/h3\u003e\n\u003cp\u003eFor the preparation of the ACY NPS, the nanoprecipitation technique was used to enhance the drug loading. In 10 mL acetone, an organic solution of a biodegradable polymer 1:1 PLGA and ACY were made. Dropwise, the organic phase was added to a 30 ml filtered pure water under continuous magnetic stirring. Centrifugation at 11,000 rpm at 4\u0026deg;C for 30 minutes was used to separate the non-encapsulated ACY. The supernatant was discarded. The final pellet was dispersed in 5 mL of water and lyophilised to get nanoparticles. Nanoparticles were stored at -20 \u0026deg;c for further use[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eCharacterisation of nanoparticle formulations\u003c/h2\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003eParticle size Surface charge and Surface morphology\u003c/h2\u003e\u003cp\u003eParticle size analyser (Nano-ZS, Malvern, Westborough, United States) was used to determine the average diameter of the particles, size distribution, polydispersity index, and Zeta potential of the prepared formulations. Nanoparticles were suspended in deionised water and bath sonicated for 30 seconds before measurement. Electrode was used to measure the zeta potential on the same instrument. The size and shape of ACPD and ACY NPs were analysed using scanning electron microscope (7610FPLUS, Jeol, Japan). Images were captured at an accelerating voltage of 5 kV, with a working distance of 7 mm and a magnification of 10,000x.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eDetermination of ACY and ACPD encapsulation in nanoparticles\u003c/h2\u003e\u003cp\u003eThe drug loading in PLGA nanoparticles (NPs) was determined by quantifying the amount of encapsulated drug per milligram of nanoparticles. A fixed quantity of freeze-dried NPs were accurately weighed and suspended in a measured volume of methanol. The suspension was kept on a rocker shaker (REMI, Mumbai, India) for 48 hours to facilitate drug release from the polymeric matrix. After incubation, the samples were centrifuged at 11,000 rpm for 20 minutes, and the resulting supernatant was collected and evaporated to dryness. The dried residue was reconstituted in an appropriate solvent and analysed using RP-HPLC to quantify the encapsulated drug. All measurements were performed in triplicate, and the mean value was reported.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eStability Study of ACY and ACPD\u003c/h2\u003e\u003cp\u003eThe stability of ACY and ACPD was evaluated in phosphate buffer solutions of pH 4.5, 5.2, and 7.4. Known amount of each drug was weighed and diluted in the respective buffers. Each sample, prepared in triplicate, was analysed by RP-HPLC to record initial readings. The drug-containing samples were incubated at 37\u0026deg;C on a rotary shaker, and at defined time intervals, they were transferred into individually labelled HPLC vials for analysis. The study was conducted over 15 days, and the graph was plotted for percentage of stable drug in Y axis and days in X axis to quantify the degradation amount of ACY and ACPD.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn Vitro\u003c/b\u003e \u003cb\u003eDrug Release Kinetics\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e drug release studies were conducted to evaluate the release of ACY from ACY-PLGA NPs and ACPD from ACPD-PLGA NPs. A known amount of lyophilised nanoparticles were accurately weighed and suspended in buffer solutions of pH 4.5 and 7.4. The samples prepared were in triplicate, incubated at 37\u0026deg;C on a rocker shaker to simulate physiological conditions. At defined time intervals, the suspensions were centrifuged at 11,000 rpm for 20 minutes, and the supernatant was collected and analysed using RP-HPLC to quantify the released drug. The nanoparticles pellet was resuspended in a fixed volume of fresh buffer and returned to the shaker for continued incubation. This process was repeated until maximum drug release was achieved. Drug release kinetics were evaluated by calculating the cumulative percentage of drug released over time (in days).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eCytotoxicity profile of test compounds in Vero cells (CPE-based)\u003c/h2\u003e\u003cp\u003eVero cells were maintained in MEM supplemented with 10% FBS and gentamycin (40 mg/mL). The cells (1.5 \u0026times; 10⁴ cells/well) were seeded in a 96-well microtiter plate and incubated at 37\u0026deg;C in a humidified 5% CO₂ environment. Test compounds were assessed at concentrations ranging from 1000 to 1.9 \u0026micro;M for cytotoxicity.\u003c/p\u003e\u003cp\u003eThe test compounds included ACY, Acyclovir-loaded nanoparticles (ACY- NPs), ACPD and ACPD loaded NPs. A stock solution of 2 mM was prepared for each test compound, followed by serial dilutions to achieve the final concentrations of 1000 \u0026micro;M, 500 \u0026micro;M, 125 \u0026micro;M, 62.5 \u0026micro;M, 31.2 \u0026micro;M, 15.6 \u0026micro;M, 7.8 \u0026micro;M, 3.9 \u0026micro;M, and 1.9 \u0026micro;M. These dilutions were prepared in MEM supplemented with 2% FBS. Each test compound concentration was added in triplicate to the Vero cell monolayer. The cells were incubated for 48 h at 37\u0026deg;C in a CO\u003csub\u003e2\u003c/sub\u003e incubator.\u003c/p\u003e\u003cp\u003eCytotoxicity was evaluated by monitoring changes in cell morphology and growth rate in cells treated with the test compounds, compared to untreated control cells. The cells were observed under inverted microscope for any morphological changes such as deterioration and detachment of cell monolayer. The plates were incubated for 48 hrs and then fixed with 70% v/v methanol and stained with 0.5% w/v crystal violet. Absorbance was measured at 490 nm using an ELISA reader (BioTek, ELx800, USA).\u003c/p\u003e\u003cp\u003eThe percentage of cytotoxicity was determined using the formula: Percentage of cell cytotoxicity = [A\u003csub\u003eC\u003c/sub\u003e \u0026ndash; A\u003csub\u003eT\u003c/sub\u003e / A\u003csub\u003eC\u003c/sub\u003e] *100; where A\u003csub\u003eC\u003c/sub\u003e = Absorbance of cell control; and A\u003csub\u003eT\u003c/sub\u003e = Absorbance of test wells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eVirus plaque reduction assay\u003c/h2\u003e\u003cp\u003eThe assay was performed using the non-cytotoxic concentrations (2-160 \u0026micro;M) of the test compounds. The dilutions of the test compounds were prepared in MEM MM. A concentration of 100 PFU (Plaque forming unit) of HSV-1 was used for the assay.\u003c/p\u003e\u003cp\u003eA confluent monolayer of Vero cells (1.25*10⁵ cells/ml) in 12-well plates were used for the assay. Media was discarded and 200 \u0026micro;l of fresh media (MEM MM) was added to all wells. The cells were inoculated with 100 PFU of HSV-1. After 1 hour of incubation at 37\u0026deg;C, virus was removed and 100 \u0026micro;L of different concentrations of the test compounds were added to the respective wells. 100 \u0026micro;L MEM MM was added to the cell and virus control wells, followed by the addition of agarose overlay media to all the wells. Post 24 hours of incubation, a second agarose overlay media with 0.3% v/v neutral red satin was added to visualize the plaques. Post 14 hours of incubation, number of plaques in each treatment group were counted using light box. Plaques were represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and analyzed statistically in comparison to virus control.\u003c/p\u003e\u003cp\u003ePercentage reduction in viral plaques was calculated using the formula: Percentage reduction in viral plaques = [(Average number of plaques in virus control - Average number of plaques in test) / Average number of plaques in virus control] *100\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eEnzymatic cleavage of ACPD to ACY\u003c/h2\u003e\u003cp\u003eFor enzymatic cleavage studies, 15 units of porcine liver esterase were dissolved in 1 mL of phosphate buffer (pH 8) to prepare the enzyme stock solution. From this, 10 \u0026micro;L (equivalent to 0.15 units) was added to ACPD solution, which was also prepared in phosphate buffer (pH 8) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Prior to enzyme addition, ACPD solution was analysed using the previously optimised RP-HPLC method developed for the quantification of ACPD. After confirming the retention time of ACPD, the enzyme was added, and the reaction mixture was incubated at 37\u0026deg;C. At 15-minute intervals, samples were analysed by RP-HPLC to monitor the enzymatic breakdown of ACPD into ACY.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eChemistry\u003c/h2\u003e\u003cp\u003eACY was reacted with benzoic acid to selectively synthesize 2-((2-amino-6-oxo-3,6-dihydro-9\u003cem\u003eH\u003c/em\u003e-purin-9-yl)methoxy)ethyl benzoate (ACPD) via \u003cem\u003eO\u003c/em\u003e-benzoylation, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as a coupling agent (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The structure of the resulting product was confirmed using FTIR, \u003csup\u003e1\u003c/sup\u003eH NMR, \u003csup\u003e13\u003c/sup\u003eC NMR, and High-Resolution Mass Spectrometry (HRMS).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe FTIR spectra of ACPD derivative exhibited characteristic absorption bands at 3447 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 3319 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponding to asymmetric and symmetric NH\u003csub\u003e2\u003c/sub\u003e stretching vibrations. A peak at 3194 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was attributed to NH stretching. The carbonyl absorptions appeared at 1713 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;O ester) and 1687 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;O pyrimidine). Additionally, the N\u0026thinsp;=\u0026thinsp;C stretching vibration was observed at 1604 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003eThe Individual FTIR spectrum of ACY and ACPD are given in Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2.\u003c/p\u003e\u003cp\u003eThis reaction selectively yielded the \u003cem\u003eO\u003c/em\u003e-benzoylated ACY, where the hydroxyl group of ACY underwent benzoylation while the primary amine (NH\u003csub\u003e2\u003c/sub\u003e) on the purine ring and the secondary amine (NH) on the pyrimidine ring remained unaffected. This selectivity was confirmed by \u003csup\u003e1\u003c/sup\u003eH NMR, which showed the disappearance of the hydroxyl (OH) proton signal between 4.40 and 5.00 ppm in the product, while the amine signals were preserved at 6.51 ppm and 10.62 ppm, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eThe \u003csup\u003e1\u003c/sup\u003eH NMR spectrum of ACPD \u003cb\u003e3\u003c/b\u003e further exhibited distinct signals for the three methylene groups at 3.82, 4.35, and 5.41 ppm, while the signal for the free primary amine appeared at 6.5 ppm. Aromatic protons, including five from the phenyl ring and one from the purine ring, resonated between 7.49 and 7.90 ppm. The purine CH proton overlapped with the phenyl protons ortho to the carbonyl group at 7.81\u0026ndash;7.87 ppm. A distinct signal for the purine NH proton appeared at 10.62 ppm. The disappearance of the hydroxyl signal around 4.7 ppm, along with the emergence of five additional aromatic proton signals, confirmed the successful \u003cem\u003eO\u003c/em\u003e-benzoylation of ACY. The \u003csup\u003e1\u003c/sup\u003eH NMR spectrum of ACY and ACPD are given in Supplementary Fig. S3 and S4.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe \u003csup\u003e13\u003c/sup\u003eC NMR spectrum displayed 13 peaks consistent with the expected structure. The highly deshielded benzoyl carbonyl carbon appeared at 166.06 ppm, while the three aliphatic methylene carbons resonated at 72.35, 66.92, and 64.02 ppm. The remaining carbon signals were observed between 117.05 and 116.09 ppm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Further, mass spectrometry was done to validate mass of the synthesised derivatives, which was found to match with theoretical value (Supplementary Fig. S5).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eRP-HPLC for Quantitative Analysis\u003c/h2\u003e\u003cp\u003eThe developed RP-HPLC method enabled the accurate and reproducible quantification of ACY and ACPD with sharp, well-resolved peaks and minimal tailing. ACY was eluted at 3.865 minutes and detected at 254 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), while ACPD showed a retention time of 8.477 minutes, detected at 245 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Simultaneous estimation of both analytes revealed retention times of 2.767 min for ACY and 8.948 min for ACPD (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003eLinearity was established across a broad concentration range for both drugs. The calibration curve for ACY (0.976\u0026ndash;200 \u0026micro;g/mL) showed coefficient of determination (R\u0026sup2;) of 0.9966 with the regression equation y\u0026thinsp;=\u0026thinsp;59045x (Supplementary Fig. S6), while ACPD exhibited strong linearity with y\u0026thinsp;=\u0026thinsp;31734x and R\u0026sup2; = 0.9986 (Supplementary Fig. S9). percent relative standard deviation (%RSD) values for both peak area and retention time remained below 1% and 0.5%, respectively, indicating excellent precision, reproducibility, and system suitability.\u003c/p\u003e\u003cp\u003eThe stacked chromatograms for ACY and ACPD are provided in Supplementary Fig. S7 and Fig. S10, respectively. Precision plots and data are included in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eand Fig. S8. The method was validated as per ICH guideline Q2(R1), confirming its robustness in terms of linearity, accuracy, and precision [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The validated RP-HPLC method offers several advantages over traditional methods, including shorter analysis times, enhanced resolution, and better adaptability to complex matrices [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003ePreparation and characterization of ACY and ACPD loaded nanoparticles\u003c/h2\u003e\u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\u003ch2\u003eSize, Shape and surface morphology\u003c/h2\u003e\u003cp\u003eThe prepared ACY-PLGA NPs had a mean particle size of 211\u0026thinsp;\u0026plusmn;\u0026thinsp;24 nm with PDI 0.477 and a mean zeta potential of -27\u0026thinsp;\u0026plusmn;\u0026thinsp;4 mV (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). ACPD-PLGA NPs had a mean particle size of 293\u0026thinsp;\u0026plusmn;\u0026thinsp;24 nm with PDI 0.230 and a mean zeta potential of -29\u0026thinsp;\u0026plusmn;\u0026thinsp;4 mV. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe average particle size of drug-free PLGA nanoparticles was found to be 249\u0026thinsp;\u0026plusmn;\u0026thinsp;43 nm and mean Zeta potential \u0026minus;\u0026thinsp;49\u0026thinsp;\u0026plusmn;\u0026thinsp;10mV as shown in Supplementary Fig. S11 and Fig. S12. SEM images revealed the size, shape and surface morphology of the prepared NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Images confirmed that the particles are spherical and are evenly distributed. The particle size measurements aligned with the results obtained from the particle size analyser, and no cracks or pores were observed, confirming the smooth surface morphology of the prepared nanoparticles.\u003c/p\u003e\u003cp\u003eThis study aimed to enhance the stability and bioavailability of ACY by encapsulating it within FDA-approved, biodegradable PLGA nanoparticles. Nanoparticles were prepared using two widely used methods. ACPD NPs and blank NPs were synthesized using the optimized single emulsion and solvent evaporation method, as described previously[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The method is typically suitable for hydrophobic drugs. However, due to the hydrophilic nature of ACY, encapsulation efficiency was suboptimal when the double emulsion solvent evaporation method was used. Consequently, the nanoprecipitation technique was adopted, which significantly improved ACY encapsulation within the nanoparticles. This highlights the importance of selecting suitable nanoformulation techniques based on the drug's physicochemical properties to ensure optimal nanoparticle performance. In our previous work, nanoparticle preparation was optimised by varying sonication time, PVA concentration, and drug amount as key formulation parameters[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePLGA degrades into non-toxic lactic and glycolic acid monomers. Polyvinyl alcohol (PVA) served as a surfactant, facilitating emulsification for encapsulation of drug into NPs. Excess PVA was removed through repeated resuspension and centrifugation in deionized water (3\u0026times; at 11,000 rpm). Various PVA concentrations (1%, 2.5%, and 5%) were tested, but 2.5% gives better particle size, stability and reduces coalescence of the particles[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eZeta potential and particle size were measured using a Zetasizer. Zeta potential, a key indicator of colloidal stability, reflects the electrostatic repulsion between particles. Values exceeding\u0026thinsp;\u0026plusmn;\u0026thinsp;30 mV typically indicate stable systems due to sufficient charge repulsion, which prevents aggregation and promotes redispersion[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The obtained values confirmed the physical stability of the optimized nanoparticle formulations.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eDrug loading into PLGA NPs\u003c/h2\u003e\u003cp\u003eThe amount of drug loaded in NPs was quantified by measuring the drug encapsulated in micrograms per milligram of nanoparticles (NPs). The optimised PLGA-ACY NPs exhibited an encapsulation of 186\u0026thinsp;\u0026plusmn;\u0026thinsp;16 \u0026micro;g/mg, while the PLGA-ACPD NPs showed a higher encapsulation amount of 271\u0026thinsp;\u0026plusmn;\u0026thinsp;12 \u0026micro;g/mg.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eStability Analysis of ACY and ACPD\u003c/h2\u003e\u003cp\u003eThe stability of ACY and ACPD was evaluated in phosphate buffer solutions at pH 7.4, 5.2, and 4.5 to mimic physiological conditions, where pH 7.4 represents blood plasma, pH 5.2 simulates the endosomal environment, and pH 4.5 corresponds to lysosomal conditions. Drug solutions were incubated at 37\u0026deg;C and analysed by RP-HPLC over 15 days. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, ACY is quite stable, approximately 96% of the drug remained at pH 7.4 after Day 15. At lower pH 5.2 (89%) and pH 4.5 (85%), the stability of ACY decreases. These findings align with previous reports on the stability of ACY, which showed significant degradation under acidic and oxidative conditions, while only minor degradation occurred in neutral and alkaline environment [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In contrast, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb shows that ACPD maintained greater stability across all pH conditions, retaining 93\u0026ndash;98% of its initial concentration over the same period. These results suggest that ACPD is more stable than ACY in both neutral and acidic conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn Vitro\u003c/b\u003e \u003cb\u003eDrug Release Kinetics\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe drug release profiles of ACY- and ACPD-loaded PLGA nanoparticles were evaluated at pH 4.5 and 7.4 to simulate lysosomal and plasma environments, respectively. ACY-loaded NPs showed a rapid initial burst, releasing approximately 68% at pH 4.5 and 73% at pH 7.4 within the first 6 hours. By 24 hours, the cumulative release reached 70% at pH 4.5 and 74% at pH 7.4, after which it remained nearly constant over the 8-day study period(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). In contrast, ACPD-loaded NPs exhibited a more gradual and sustained release, with 16% at pH 4.5 and 20% at pH 7.4 in the first 6 hours, followed by 35% at pH 4.5 and 42% at pH 7.4 by 24 hours. The cumulative release extended to 86% at pH 4.5 and 91% at pH 7.4 over 28 days(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eThe faster release of ACY can be attributed to its hydrophilic nature, which facilitates rapid diffusion through the aqueous polymer matrix [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In contrast, the slower release of ACPD reflects its hydrophobicity and stronger interaction with the PLGA network. PLGA nanoparticles, owing to their biocompatibility and biodegradability, provide a protective environment and supports controlled and sustained drug release while minimising premature degradation [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eACPD, synthesised from ACY via ester bond formation, undergoes hydrolytic cleavage to release ACY under acidic conditions more than at neutral pH. \u003cem\u003eIn vitro\u003c/em\u003e studies of ACPD-loaded PLGA nanoparticles revealed both sustained release of ACPD and its hydrolytic cleavage to ACY over time. The formation of ACY from ACPD was confirmed by the RP-HPLC method developed specifically for ACPD quantification. The cleavage was more evident at pH 4.5 than at pH 7.4, consistent with proton-catalysed hydrolysis of the O-benzoyl ester bond under acidic conditions [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. RP-HPLC chromatograms confirming ACY release from ACPD are provided in Supplementary Fig. S13.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eCytotoxicity profile of test compounds in Vero cells\u003c/h2\u003e\u003cp\u003eThe cytotoxicity profiles of ACY, ACPD, ACY- NPs, and ACPD- NPs were assessed in Vero cells using crystal violet staining. The 50% cytotoxic concentration (CC50) values were determined to be 1230 \u0026micro;M for ACY, 960 \u0026micro;M for ACY-NPs, 800 \u0026micro;M for ACPD, and 1000 \u0026micro;M for ACPD-NPs, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea. All nanoparticle formulations exhibited no cytotoxicity up to a concentration of 160 \u0026micro;M, while ACY and ACPD demonstrated minimal cytotoxic effects. The cytotoxicity ACY is given in Supplementary Fig. S14.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eAntiviral activity by plaque reduction\u003c/h2\u003e\u003cp\u003eThe percentage reduction in viral growth at varying concentrations of the test compounds is depicted in (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). The results are presented as percentage reduction in plaque count relative to the drug concentration. Antiviral activity was evaluated for non-cytotoxic concentrations (2 \u0026micro;M and 160 \u0026micro;M) of the test compounds. At 2 \u0026micro;M, none of the compounds exhibited significant antiviral effect, except ACY-NPs which showed 23% inhibition. Further, ACY- NPs demonstrated a notable reduction in plaques, starting from 5 \u0026micro;M, with complete inhibition (100%) at 20 \u0026micro;M. This was better than the standard, ACY, which showed 100% inhibition at 160 \u0026micro;M. In contrast, ACPD and ACPD-NPs exhibited minimal antiviral activity compared to both ACY and ACY-NPs, achieving only 50% plaque reduction at the highest concentration (160 \u0026micro;M) tested. These findings were further validated by esterase enzyme activity assays, which were conducted to analyse the observed differences.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eACPD to ACY through enzymatic cleavage\u003c/h2\u003e\u003cp\u003eAt the beginning of the experiment (time 0 minutes), ACPD concentration was at its maximum. Over time, esterase activity gradually cleaved the ester bond in ACPD, yielding the active drug ACY. The retention times for ACY and ACPD were 2.76 minutes and 8.49 minutes, respectively, and by 90 minutes, most of ACPD had been cleaved into ACY, as shown in Supplementary Fig. S15. In the plaque reduction assay, ACPD showed only 50% inhibition at 160 \u0026micro;M, which was attributed to delayed cleavage due to the absence of esterase enzyme in Vero cells and in the culture medium. Although ester bonds can undergo hydrolytic cleavage, the process is very slow. To establish the proof of concept of conversion of ACPD to ACY through esterase enzyme, \u003cem\u003ein vitro\u003c/em\u003e experiment using porcine liver esterase was performed, which successfully cleaved ACPD into ACY and verified by RP-HPLC. These findings highlight the critical role of corneal esterase in converting any esterified prodrug to active drug is reported previously [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFrom our findings, it is suggested that presence of esterase enzymes in \u003cem\u003ein-vivo\u003c/em\u003e can facilitate ACPD cleavage, potentially enhance the therapeutic efficacy of ACPD. Esterase-mediated cleavage of ester bonds is critical for the breakdown of prodrug to drug and controlled release of active pharmaceutical ingredients in ocular drug delivery systems[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Understanding esterase activity in ocular tissues is essential for designing prodrugs and polymeric carriers that depend on enzymatic activation for therapeutic efficacy. This mechanism enables site-specific drug release and reduces systemic associated side effects. For example, latanoprost, a prostaglandin analogue, relies on corneal epithelial esterase to cleave its ester bonds and release the active compound, as demonstrated in reported studies [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIn the present study, the ACY NPs were formed by applying a simple and efficient nanoprecipitation method. ACPD was synthesized and encapsulated successfully in PLGA nanoparticles using single emulsion solvent evaporation method. The formulated ACY and ACPD-loaded PLGA NPs exhibited almost uniform particle size, smooth surface morphology, and good surface charge. The formulated NPs are found to be appropriate for treating HSK. ACY NPs demonstrated significantly higher efficacy in inhibiting HSV growth compared to free ACY in Vero cells. ACPD-NPs, showed a more stable and controlled release profile across various pH conditions. Esterase enzyme assay confirmed the cleavage of ACPD to ACY and these findings suggest that the presence of esterase enzymes in animals can facilitate the cleavage of ACPD, potentially enhancing the therapeutic efficacy of ACPD. This nanoparticle formulation presents an efficient delivery system for the treatment of ocular viral infections. Future \u003cem\u003ein vivo\u003c/em\u003e studies will further confirm the effectiveness of this nanocarrier-based drug delivery system to treat HSK.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS.S.H:\u003c/strong\u003e Data curation, Investigation, Formal analysis, Writing \u0026ndash; original draft \u0026amp; editing.\u0026nbsp;\u003cstrong\u003e\u0026nbsp;D.V. P:\u003c/strong\u003e Data curation, Investigation, Formal analysis, Writing \u0026ndash; original draft \u0026amp; editing.\u003cstrong\u003e\u0026nbsp;S.L.G:\u0026nbsp;\u003c/strong\u003eData curation, writing and editing.\u003cstrong\u003e\u0026nbsp;K.K:\u0026nbsp;\u003c/strong\u003eData curation, writing and editing.\u003cstrong\u003e\u0026nbsp;P.P.M:\u0026nbsp;\u003c/strong\u003eData curation, writing and editing.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Y.N.N: \u0026nbsp;\u003c/strong\u003eData curation, writing and editing.\u003cstrong\u003e\u0026nbsp;B.R.G\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eConceptualization, Supervision, writing \u0026ndash; review, editing and final approval of the manuscript. All the authors read the final manuscript and approved for the publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Manipal Center for Infectious Diseases (MAC ID) for awarding intramural grant for this research work. \u0026nbsp;We thank Manipal Institute of Technology, Manipal and Manipal Institute of Virology, Manipal for allowing to conduct this research. We thank Manipal Academy of Higher Education, Manipal for proving Dr. TMA Pai scholarship to D.V.P.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo potential conflict of interest was reported by the author(s).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is presented in the main manuscript and supplementary information.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eManipal Center for Infectious Diseases (MAC ID), an intramural grant from Manipal Academy of Higher Education (MAHE), Manipal.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHerpes simplex virus n.d. accessed April 27, (2025). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.who.int/news-room/fact-sheets/detail/herpes-simplex-virus#\u003c/span\u003e\u003cspan address=\"https://www.who.int/news-room/fact-sheets/detail/herpes-simplex-virus#\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcCormick, I. et al. \u003cem\u003eOphthalmic Epidemiol.\u003c/em\u003e ;\u003cb\u003e29\u003c/b\u003e:353. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/09286586.2021.1962919\u003c/span\u003e\u003cspan address=\"10.1080/09286586.2021.1962919\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMaroui, M. A. et al. Latency Entry of Herpes Simplex Virus 1 Is Determined by the Interaction of Its Genome with the Nuclear Environment. \u003cem\u003ePLoS Pathog\u003c/em\u003e. \u003cb\u003e12\u003c/b\u003e, e1005834. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/JOURNAL.PPAT.1005834\u003c/span\u003e\u003cspan address=\"10.1371/JOURNAL.PPAT.1005834\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAl-Dujaili, L. J. et al. Ocular herpes simplex virus: how are latency, reactivation, recurrent disease and therapy interrelated? \u003cem\u003eFuture Microbiol.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e, 877. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2217/FMB.11.73\u003c/span\u003e\u003cspan address=\"10.2217/FMB.11.73\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKimberlin, D. W. \u0026amp; Whitley, R. J. Antiviral therapy of HSV-1 and \u0026ndash;\u0026thinsp;2. \u003cem\u003eHum. Herpesviruses: Biology Therapy Immunoprophylaxis\u003c/em\u003e \u003cb\u003e2007\u003c/b\u003e:1153\u0026ndash;1174. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1017/CBO9780511545313.065\u003c/span\u003e\u003cspan address=\"10.1017/CBO9780511545313.065\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePoole, C. L. \u0026amp; James, S. H. Antiviral Therapies for Herpesviruses: Current Agents and New Directions. \u003cem\u003eClin. Ther.\u003c/em\u003e \u003cb\u003e40\u003c/b\u003e, 1282. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.CLINTHERA.2018.07.006\u003c/span\u003e\u003cspan address=\"10.1016/J.CLINTHERA.2018.07.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLeBlanc, R. A., Pesnicak, L., Godleski, M. \u0026amp; Straus, S. E. The Comparative Effects of Famciclovir and Valacyclovir on Herpes Simplex Virus Type 1 Infection, Latency, and Reactivation in Mice. \u003cem\u003eJ. Infect. Dis.\u003c/em\u003e \u003cb\u003e180\u003c/b\u003e, 594\u0026ndash;599. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1086/314962\u003c/span\u003e\u003cspan address=\"10.1086/314962\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1999).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHawthorne, K. M., Dana, R. \u0026amp; Chodosh, J. Delayed Type Hypersensitivity in the Pathogenesis of Recurrent Herpes Stromal Keratitis. \u003cem\u003eSemin Ophthalmol.\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e, 246\u0026ndash;250. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3109/08820538.2011.588659\u003c/span\u003e\u003cspan address=\"10.3109/08820538.2011.588659\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStanfield, B. \u0026amp; Kousoulas, K. G. Herpes Simplex Vaccines: Prospects of Live-attenuated HSV Vaccines to Combat Genital and Ocular infections. \u003cem\u003eCurr. Clin. Microbiol. Rep.\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e, 125. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/S40588-015-0020-4\u003c/span\u003e\u003cspan address=\"10.1007/S40588-015-0020-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVan Wagoner, N., Qushair, F. \u0026amp; Johnston, C. Genital Herpes Infection: Progress and Problems. \u003cem\u003eInfect. Dis. Clin. North. Am.\u003c/em\u003e \u003cb\u003e37\u003c/b\u003e, 351\u0026ndash;367. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.IDC.2023.02.011\u003c/span\u003e\u003cspan address=\"10.1016/J.IDC.2023.02.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlvaro, P. M., Sitar, D. S. \u0026amp; Aoki, F. Y. Comparative bioavailability of acyclovir from oral valacyclovir and acyclovir in patients treated for recurrent genital herpes simplex virus infection. \u003cem\u003eCan. J. Clin. Pharmacol.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 207\u0026ndash;211 (2001).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDonalisio, M. et al. Acyclovir-Loaded Chitosan Nanospheres from Nano-Emulsion Templating for the Topical Treatment of Herpesviruses Infections. \u003cem\u003ePharmaceutics\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 46. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/PHARMACEUTICS10020046\u003c/span\u003e\u003cspan address=\"10.3390/PHARMACEUTICS10020046\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFrejborg, F., Kalke, K. \u0026amp; Hukkanen, V. Current landscape in antiviral drug development against herpes simplex virus infections 2022. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/SMMD.20220004\u003c/span\u003e\u003cspan address=\"10.1002/SMMD.20220004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYadav, K. S. et al. Microemulsions for enhancing drug delivery of hydrophilic drugs: Exploring various routes of administration. \u003cem\u003eMed. Drug Discov\u003c/em\u003e. \u003cb\u003e20\u003c/b\u003e, 100162. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.MEDIDD.2023.100162\u003c/span\u003e\u003cspan address=\"10.1016/J.MEDIDD.2023.100162\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFreeman, D. J., Sheth, N. V. \u0026amp; Spruance, S. L. Failure of topical acyclovir in ointment to penetrate human skin. \u003cem\u003eAntimicrob. Agents Chemother.\u003c/em\u003e \u003cb\u003e29\u003c/b\u003e, 730\u0026ndash;732. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AAC.29.5.730\u003c/span\u003e\u003cspan address=\"10.1128/AAC.29.5.730\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1986).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSoul-Lawton, J. et al. Absolute bioavailability and metabolic disposition of valaciclovir, the L-valyl ester of acyclovir, following oral administration to humans. \u003cem\u003eAntimicrob. Agents Chemother.\u003c/em\u003e \u003cb\u003e39\u003c/b\u003e, 2759\u0026ndash;2764. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AAC.39.12.2759\u003c/span\u003e\u003cspan address=\"10.1128/AAC.39.12.2759\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1995).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHeikkinen, E. M. et al. Esterase activity in porcine and albino rabbit ocular tissues. \u003cem\u003eEur. J. Pharm. Sci.\u003c/em\u003e \u003cb\u003e123\u003c/b\u003e, 106\u0026ndash;110. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.EJPS.2018.07.034\u003c/span\u003e\u003cspan address=\"10.1016/J.EJPS.2018.07.034\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHammid, A. et al. Activity and Expression of Carboxylesterases and Arylacetamide Deacetylase in Human Ocular Tissues. \u003cem\u003eDrug Metab. Dispos.\u003c/em\u003e \u003cb\u003e50\u003c/b\u003e, 1483\u0026ndash;1492. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1124/DMD.122.000993\u003c/span\u003e\u003cspan address=\"10.1124/DMD.122.000993\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePerez, C., Daniel, K. B. \u0026amp; Cohen, S. M. Evaluating Prodrug Strategies for Esterase-Triggered Release of Alcohols. ChemMedChem. ;8:1662. (2013). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/CMDC.201300255\u003c/span\u003e\u003cspan address=\"10.1002/CMDC.201300255\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMahboobian, M. M., Mohammadi, M. \u0026amp; Mansouri, Z. Development of thermosensitive in situ gel nanoemulsions for ocular delivery of acyclovir. \u003cem\u003eJ. Drug Deliv Sci. Technol.\u003c/em\u003e 55. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.JDDST.2019.101400\u003c/span\u003e\u003cspan address=\"10.1016/J.JDDST.2019.101400\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDonalisio, M. et al. Acyclovir-Loaded Chitosan Nanospheres from Nano-Emulsion Templating for the Topical Treatment of Herpesviruses Infections. \u003cem\u003ePharmaceutics\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 46. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/PHARMACEUTICS10020046\u003c/span\u003e\u003cspan address=\"10.3390/PHARMACEUTICS10020046\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMerkli, A., Tabatabay, C., Gurny, R. \u0026amp; Heller, J. Biodegradable polymers for the controlled release of ocular drugs. \u003cem\u003eProg Polym. Sci.\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e, 563\u0026ndash;580. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0079-6700(97)00048-8\u003c/span\u003e\u003cspan address=\"10.1016/S0079-6700(97)00048-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1998).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang, Y. et al. Nanotechnology\u0026rsquo;s frontier in combatting infectious and inflammatory diseases: prevention and treatment. \u003cem\u003eSignal. Transduct. Target. Therapy 2024\u003c/em\u003e. \u003cb\u003e9\u003c/b\u003e, 1. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41392-024-01745-z\u003c/span\u003e\u003cspan address=\"10.1038/s41392-024-01745-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBlecher, K., Nasir, A. \u0026amp; Friedman, A. The growing role of nanotechnology in combating infectious disease. \u003cem\u003eVirulence\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e, 395\u0026ndash;401. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4161/VIRU.2.5.17035\u003c/span\u003e\u003cspan address=\"10.4161/VIRU.2.5.17035\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHakkimane, S. S., Shenoy, V. P., Gaonkar, S. L., Bairy, I. \u0026amp; Guru, B. R. Antimycobacterial susceptibility evaluation of rifampicin and isoniazid benz-hydrazone in biodegradable polymeric nanoparticles against Mycobacterium tuberculosis H37Rv strain. \u003cem\u003eInt. J. Nanomed.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 4303\u0026ndash;4318. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2147/IJN.S163925\u003c/span\u003e\u003cspan address=\"10.2147/IJN.S163925\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePraveena, J. \u0026amp; Guru, B. R. Simultaneous estimation of paclitaxel and curcumin in nano-formulation: Stability analysis of drugs, optimization and validation of HPLC method. \u003cem\u003eJ. Appl. Pharm. Sci.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e,, 071\u0026ndash;83. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7324/JAPS.2021.110308\u003c/span\u003e\u003cspan address=\"10.7324/JAPS.2021.110308\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHakkimane, S. S. \u0026amp; Guru, B. R. Nano formulation analysis: Analytical method development of isoniazid and simultaneous estimation of antitubercular drugs isoniazid and rifampicin by reverse phase high pressure liquid chromatography. \u003cem\u003eAsian J. Pharm. Clin. Res.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 330\u0026ndash;335. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.22159/AJPCR.2017.V10I5.17582\u003c/span\u003e\u003cspan address=\"10.22159/AJPCR.2017.V10I5.17582\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGovender, T., Stolnik, S., Garnett, M. C., Illum, L. \u0026amp; Davis, S. S. PLGA nanoparticles prepared by nanoprecipitation: drug loading and release studies of a water soluble drug. \u003cem\u003eJ. Control Release\u003c/em\u003e. \u003cb\u003e57\u003c/b\u003e, 171\u0026ndash;185. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0168-3659(98)00116-3\u003c/span\u003e\u003cspan address=\"10.1016/S0168-3659(98)00116-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1999).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMuszalska-Kolos, I., Lesniewska-Kowiel, M. A., Plewa, S. \u0026amp; Klupczyńska, A. Tricyclic Derivative of Acyclovir and Its Esters in Relation to the Esters of Acyclovir Enzymatic Stability: Enzymatic Stability Study. Molecules 2020;25. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/MOLECULES25092156\u003c/span\u003e\u003cspan address=\"10.3390/MOLECULES25092156\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eINTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE ICH HARMONISED TRIPARTITE GUIDELINE VALIDATION OF ANALYTICAL PROCEDURES. TEXT AND METHODOLOGY Q2(R1) n.d.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAboelezz, A., Kharouba, M. \u0026amp; Mahmoud, S. H. A simple method for the determination of acyclovir concentrations in human plasma using high-performance liquid chromatography. \u003cem\u003eFuture J. Pharm. Sci. 2024\u003c/em\u003e. \u003cb\u003e10\u003c/b\u003e, 1. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/S43094-024-00649-7\u003c/span\u003e\u003cspan address=\"10.1186/S43094-024-00649-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePraveena, J., Hakkimane, S. S. \u0026amp; Guru, B. R. Synergistic effect of Paclitaxel and Curcumin in nano-formulations on U87 and A549 cancer cell lines. \u003cem\u003eJ. Appl. Pharm. Sci.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e,, 031\u0026ndash;47. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7324/JAPS.2021.120204\u003c/span\u003e\u003cspan address=\"10.7324/JAPS.2021.120204\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHakkimane, S. S., Shenoy, V. P., Gaonkar, S. L., Bairy, I. \u0026amp; Guru, B. R. Antimycobacterial susceptibility evaluation of rifampicin and isoniazid benz-hydrazone in biodegradable polymeric nanoparticles against Mycobacterium tuberculosis H37Rv strain. \u003cem\u003eInt. J. Nanomed.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 4303\u0026ndash;4318. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2147/IJN.S163925\u003c/span\u003e\u003cspan address=\"10.2147/IJN.S163925\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePinto, J., Ahmad, M. \u0026amp; Guru, B. R. Enhancing the efficacy of fluocinolone acetonide by encapsulating with PLGA nanoparticles and conjugating with linear PEG polymer. \u003cem\u003eJ. Biomater. Sci. Polym. Ed.\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e, 1188\u0026ndash;1211. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/09205063.2019.1625524\u003c/span\u003e\u003cspan address=\"10.1080/09205063.2019.1625524\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSegets, D. et al. Experimental and theoretical studies of the colloidal stability of nanoparticles-a general interpretation based on stability maps. \u003cem\u003eACS Nano\u003c/em\u003e. \u003cb\u003e5\u003c/b\u003e, 4658\u0026ndash;4669. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/NN200465B\u003c/span\u003e\u003cspan address=\"10.1021/NN200465B\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSinha, V. R., Monika, Trehan, A., Kumar, M., Singh, S. \u0026amp; Bhinge, J. R. Stress studies on acyclovir. \u003cem\u003eJ. Chromatogr. Sci.\u003c/em\u003e \u003cb\u003e45\u003c/b\u003e, 319\u0026ndash;324. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/CHROMSCI/45.6.319\u003c/span\u003e\u003cspan address=\"10.1093/CHROMSCI/45.6.319\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJain, A. et al. Peptide and protein delivery using new drug delivery systems. \u003cem\u003eCrit. Rev. Ther. Drug Carrier Syst.\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e, 293\u0026ndash;329. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1615/CRITREVTHERDRUGCARRIERSYST.2013006955\u003c/span\u003e\u003cspan address=\"10.1615/CRITREVTHERDRUGCARRIERSYST.2013006955\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMakadia, H. K. \u0026amp; Siegel, S. J. Poly Lactic-co-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier. \u003cem\u003ePolym. (Basel)\u003c/em\u003e. \u003cb\u003e3\u003c/b\u003e, 1377. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/POLYM3031377\u003c/span\u003e\u003cspan address=\"10.3390/POLYM3031377\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlsaab, H. O. et al. PLGA-Based Nanomedicine: History of Advancement and Development in Clinical Applications of Multiple Diseases. \u003cem\u003ePharmaceutics\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 2728. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/PHARMACEUTICS14122728\u003c/span\u003e\u003cspan address=\"10.3390/PHARMACEUTICS14122728\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang, F. et al. The Hydrolysis of Diclofenac Esters: Synthetic Prodrug Building Blocks for Biodegradable Drug-Polymer Conjugates. \u003cem\u003eJ. Pharm. Sci.\u003c/em\u003e \u003cb\u003e105\u003c/b\u003e, 773\u0026ndash;785. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jps.24665\u003c/span\u003e\u003cspan address=\"10.1002/jps.24665\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKuny, C. V., Bowen, C. D., Renner, D. W., Johnston, C. M. \u0026amp; Szpara, M. L. In vitro evolution of herpes simplex virus 1 (HSV-1) reveals selection for syncytia and other minor variants in cell culture. \u003cem\u003eVirus Evol.\u003c/em\u003e 6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/VE/VEAA013\u003c/span\u003e\u003cspan address=\"10.1093/VE/VEAA013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAkkurt Arslan, M. et al. Profiling tear film enzymes reveals major metabolic pathways involved in the homeostasis of the ocular surface. \u003cem\u003eSci. Rep. 2023\u003c/em\u003e. \u003cb\u003e13\u003c/b\u003e, 1. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-023-42104-2\u003c/span\u003e\u003cspan address=\"10.1038/s41598-023-42104-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTripathy, K., Patel, P., Geetha, R., Latanoprost \u0026amp; XPharm \u003cem\u003eCompr. Pharmacol. Ref.\u003c/em\u003e :1\u0026ndash;4. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/B978-008055232-3.62015-X\u003c/span\u003e\u003cspan address=\"10.1016/B978-008055232-3.62015-X\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Acyclovir, Acyclovir prodrug, nanoparticles, poly (lactic-co-glycolic acid), Herpes simplex virus","lastPublishedDoi":"10.21203/rs.3.rs-7427697/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7427697/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHerpes simplex virus-1 (HSV-1) is a widespread, recurrent infection that causes herpes simplex keratitis (HSK), an ocular disease that can lead to vision impairment. Conventional acyclovir (ACY) therapy is limited by poor bioavailability, rapid clearance, and frequent dosing. We developed an esterified acyclovir prodrug (ACPD) formulated as biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) to improve stability, enable sustained release, and enhance antiviral activity against HSV-1. ACPD was synthesized and characterized by FTIR, NMR, and mass spectrometry. Its increased hydrophobicity improved encapsulation in PLGA relative to ACY. ACY-NPs and ACPD-NPs were prepared by nanoprecipitation and single-emulsion solvent evaporation method, respectively. Optimized NPs measured 200\u0026ndash;300 nm with polydispersity indices of 0.23\u0026ndash;0.47 and zeta potentials of \u0026minus;\u0026thinsp;27 to \u0026minus;\u0026thinsp;29 mV; SEM showed uniform particles. ACPD-NPs provided sustained release for 28 days. Cytotoxicity in Vero cells yielded CC50 values of 960 \u0026micro;M (ACY-NPs) and 1000 \u0026micro;M (ACPD-NPs). In HSV-1-infected Vero cells, ACY-NPs exhibited greater antiviral efficacy than free ACY, whereas ACPD and ACPD-NPs showed minimal plaque reduction because the \u003cem\u003ein vitro\u003c/em\u003e media lacked esterase to convert ACPD to ACY; with esterase, cleavage was confirmed by RP-HPLC. Corneal tissues contain esterase, \u003cem\u003ein vivo\u003c/em\u003e activation may improve ACPD efficacy and bioavailability and overall therapeutic performance.\u003c/p\u003e","manuscriptTitle":"Biodegradable Polymeric Nano formulation of Acyclovir and Acyclovir prodrug for Enhanced Therapeutic Efficacy Against Herpes Simplex Virus Infections","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 17:24:34","doi":"10.21203/rs.3.rs-7427697/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-13T17:52:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-09T22:22:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"88551252014438855452448866746394228628","date":"2026-03-31T14:20:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-23T12:33:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"115210532391953968887969530167641134960","date":"2026-01-14T08:56:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-19T05:19:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"129706098203154579194457099791296445888","date":"2025-09-15T10:56:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-10T10:14:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-10T10:11:45+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-04T03:23:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-30T08:44:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-08-30T08:40:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"32da99d0-1c26-48ab-a51d-b801b0257532","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":54731182,"name":"Biological sciences/Biotechnology"},{"id":54731183,"name":"Biological sciences/Drug discovery"},{"id":54731184,"name":"Biological sciences/Microbiology"},{"id":54731185,"name":"Physical sciences/Nanoscience and technology"}],"tags":[],"updatedAt":"2026-05-08T19:24:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-17 17:24:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7427697","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7427697","identity":"rs-7427697","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}