Comparative Study on Drug Encapsulation and Release Kinetics in Extracellular Vesicles Loaded with Snake Venom L - amino acid oxidase

Comparative Study on Drug Encapsulation and Release Kinetics in Extracellular Vesicles Loaded with Snake Venom L - amino acid oxidase | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative Study on Drug Encapsulation and Release Kinetics in Extracellular Vesicles Loaded with Snake Venom L - amino acid oxidase Divya Ramesh, Shankar M Bakkannavar, Vinutha R Bhat, K Sreedhara Ranganath Pai, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6004383/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 May, 2025 Read the published version in BMC Pharmacology and Toxicology → Version 1 posted 5 You are reading this latest preprint version Abstract Background This study aimed to evaluate the potential of plasma-derived extracellular vesicles (EVs) as drug delivery carriers by employing two drug-loading techniques: coincubation and freeze‒thaw cycles. Methods EVs isolated via the polyethylene glycol (PEG) precipitation method were characterized via nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The size of the particles was 200.1 ± 66.6 nm. The isolated vesicles were loaded with 1000 µg/ml snake venom L amino acid oxidase (SVLAAO) via the coincubation method and subjected to freeze‒thaw cycles to prepare a novel formulation. The encapsulation efficiency (EE) of the loaded EVs was analysed at 30 and 60 minutes, and in vitro drug release profiles were evaluated for both methods and kinetic model for the same was determined. Results The coincubation method achieved an EE of 58.08 ± 0.060% after 60 minutes, which was greater than that of the freeze‒thaw method (55.80 ± 0.060%). Drug release studies demonstrated that 93% of the drug was released in 8.5 hours by the coincubation method, whereas the freeze‒thaw method resulted in faster release (99% in 6.5 hours) due to membrane disruption. The best fit value (R 2 ) was highest for zero order kinetics model. Conclusion In conclusion, the coincubation method preserves EV membrane integrity, enabling sustained drug release, making it a promising strategy for targeted drug delivery applications. This study highlights plasma-derived EVs as innovative carriers for therapeutic delivery. Extracellular vesicles Snake venom L amino acid oxidase Encapsulation efficiency Drug delivery Nanoparticles Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. INTRODUCTION Drug delivery involves the formulation of pharmacologically active substances for desired therapeutic outcomes when these substances are administered. Hence, it has been considered a fast-emerging field in nanoscience in recent decades( 1 ). Although there has been a marked increase in the development of nano delivery tools, certain disadvantages exist, such as off-target delivery, lower efficacy, and reactions with other substances, thus making the system less effective( 2 ). Thus, there is a need to develop a novel, innovative drug delivery system that increases the efficacy of the reaction, reduces the off-target distribution and causes the controlled release of the drug. In recent years, the development of synthetic nanocarriers for drug delivery has tremendously increased. Liposomes, dendrimers, micelles, nanocapsules, etc., constitute the most common types of synthetic drug carriers ( 3 ). Among these, liposomes, are considered the most versatile and are used in various nanoformulations( 4 ). Despite their numerous advantages, and its therapeutic efficiency, there are many disadvantages, such as off-target delivery, increased clearance from the reticuloendothelial system, and increased accumulation in the liver and other organs, thus limiting its availability in the target site. In addition to lipid-based carriers, other nanocarriers called extracellular vesicles (EVs) derived from cells have gained increased importance in the last few years. EVs are tiny lipid-bound particles that are important regulators of many pathophysiological reactions. Recent studies explain the similarities between liposomes and EVs as drug delivery systems and the presence of phospholipids. However, the difference lies in the specific key markers on the EVs, which aid in targeting specific areas( 5 , 6 ). EVs are classified into different types, such as ectosomes, exosomes, microvesicles, and apoptotic bodies. Although EVs are derived from different sources, they are classified according to their biogenesis pathway( 7 ). Exosomes are specific intraluminal vesicles formed during the maturation and development of multivesicular endosomes. The size of the exosomes is generally less than 200 nm. These tiny vesicles contain many surface markers, such as CD9, CD37 and CD63, which help with protein translocation and sorting( 8 – 10 ).Microvesicles or microparticles are EVs originating from the cell outer membrane. Apoptotic bodies (ApoBDs) are EVs released by cells undergoing programmed cell death or apoptosis. EVs are present in almost all biological fluids, such as blood plasma, serum, urine, breast milk, and amniotic fluid( 11 ). The most commonly used source for EV biosynthesis is blood plasma, which is the most stable and biocompatible with the body's physiological conditions. Plasma-derived EVs enhance the transport of drugs through biological barriers( 12 ). For these reasons, plasma-derived EVs are considered the best vehicles for cargo transportation. In some instances, plasma-derived EVs are involved in several pathological conditions and, hence, can be used as biomarkers for various diseases( 13 ). Hence, EVs derived from cells or any biological fluid can be used as therapeutic agents by loading the necessary drugs into them and targeting the same site of interest. EVs are believed to be involved in important physiological processes, such as the differentiation of cells( 14 ). The primary functions of EVs include transporting various substances to target organs. These vesicles act as vehicles to transport substances such as RNA species, DNA and various proteins( 15 ). All these properties of EVs make them among the most efficient and versatile drug carriers. The loading of the EVs is based on the properties of the cargo to be loaded. Passive loading is the easiest method for loading drugs into EVs. This phenomenon occurs when the cells are cocultured with the drug of interest (cargo to be loaded) for a specific time. In this method, the cells incorporate the cargo and package it into the EVs during the latter formation. As this method is simple to perform, the loading efficiency is very low compared with that of active loading. Moreover, it is difficult to control the amount of drug loaded. The passive incubation method involves coincubation of the drug/cargo to be loaded with the EVs at room temperature for a specified period (usually 30 minutes to 60 minutes). Compared with the other techniques, coincubation is the most straightforward loading strategy. Passive loading occurs when the cargo concentration in the outer environment is high. Whereas Active loading occurs against the concentration gradient and results in the formation of pores in the EV membrane, thus resulting in efficient loading( 16 ). The active method involves loading the cargo into the EV by disrupting its membrane( 17 ). The active loading methods include electroporation, sonication, saponin treatment, freeze‒thaw cycles, etc. Compared to higher loading efficiency of electroporation and sonication, freeze-thaw cycles have intermediate loading efficiency. Loading by freeze thaw cycles provide intermediate loading efficiency that involves disruption of the EV membrane by rapid freezing (-80°C) and thawing at room temperature (37°C) for a specified amount of time. This method is repeated for three continuous cycles so that the EV membrane is damaged, and the cargo is loaded successfully( 18 ). Here ,membrane damage is not permanent and occurs due to the formation of ice crystals on the membrane so that the water-soluble cargo can enter the EVs( 19 ).Thus ,compared with passive loading techniques, active loading is complex, as it involves a risk of rapid EV membrane disruption, which may damage the functions of EVs( 20 ). The choice of loading method depends on the characteristics of the drug to be loaded, the physiological conditions under which the drug must act, and the desired therapeutic efficiency of the cargo( 21 ). The optimum method for drug loading should provide maximum loading efficiency with less damage to the EV. This research highlights the potential of plasma-derived EVs as effective drug carriers. This study emphasized the loading of snake venom-derived L amino acid oxidase (SVLAAO) into extracellular vesicles isolated from plasma. SVLAAO has emerged as a potential therapeutic since it possesses antimicrobial and anticancer properties. LAAO from Bathrops snake venom can inhibit biofilm formation by Escherichia coli and Staphylococcus aureus by altering the morphology of these species, resulting in antimicrobial properties( 22 ). Several other studies have shown that SVLAAO can induce cell death, thus inhibiting cancer cell metastasis( 23 ). This property of the snake venom L amino acid oxidase makes it potentially therapeutic against cancer. This study involves loading snake venom-derived L-amino acid oxidase into plasma-derived extracellular vesicles via two different methods: coincubation and freeze‒thaw cycles. Because of its proven cytotoxic and apoptotic effects on the C6 rat glioma cell line in vitro, snake venom L-amino acid oxidase (SV-LAAO) was selected as the appropriate drug to be loaded into extracellular vesicles (EVs). This discovery, which was made possible by a pilot study, offered a strong rationale for additional research into the possible therapeutic use of SV-LAAO-loaded EVs in the treatment of glioblastoma. The outcome was evaluated via encapsulation efficiency and drug release studies. Moreover, this study evaluates the impact of two different loading techniques into the human plasma derived extracellular vesicles and was evaluated by the extent of the drug release, thus checking the efficiency of human plasma derived EVs as a potent drug carrier that will be targeted to glioblastoma multiforme (brain tumour) in vivo (Wistar rats). 2. MATERIALS AND METHODS 2.1 MATERIALS Snake venom L amino acid oxidase was purchased from Sigma Aldrich (Cat no: A5147); human blood samples were collected from healthy volunteers after institutional ethical approval was obtained from Kasturba Hospital Manipal (IEC no: 68/2022). Institutional biosafety committee clearance products were obtained from the School of Life Sciences Manipal in August 2022, including polyethylene glycol (w/v, 6000, Sigma‒Aldrich 81260), phosphate buffer saline (pH 7.4,6.4), and dialysis membranes. A schematic representation of the methodology is depicted in Fig. 1. 2.2 METHODS 2.2.1 Isolation of Extracellular Vesicles from Human Plasma: Extracellular vesicles were isolated from the plasma of healthy individuals via the polyethylene glycol precipitation method ( 24 , 25 ). Informed consent was obtained from all individual participants included in the study. Five milliliters of blood was collected from healthy individuals in an EDTA-coated vacutainer, and the plasma was separated by centrifugation at 2500xg for 20 minutes. The separated plasma was centrifuged at 2500xg for 20 minutes to remove RBCs and other debris. The collected supernatant was treated with 2X 12% polyethylene glycol (PEG) and incubated overnight at 4°C. The mixture was centrifuged at 3300xg for 1 hour at 4°C to separate the pellet and the supernatant. The resulting pellet was resuspended in sterile PBS (1–3 ml), an equal amount of 2X 5% PEG was added, and the mixture was incubated at 4°C for 1 hour. In the final step, the mixture was centrifuged at 3300xg for 1 hour at 4°C, and the obtained pellet (EVs) was resuspended in 200 µl of sterile PBS (pH 7.4). 2.2.2 Characterization of Extracellular vesicles The plasma-derived EVs were characterized according to the MISEV 2023 guidelines( 26 ). The concentration of the isolated EVs and their size were determined via nanoparticle tracking analysis (NTA) via a NanoSight LM10 (Malvern Instruments, UK). EV samples resuspended in PBS were vortexed and diluted 1:1000 to detect particles within the optimum detection limit. The experiments were performed three times to identify and confirm the EVs isolated from the plasma. Morphology of the EVs The structure of the EVs was determined via transmission electron microscopy (TEM). TEM was performed via the negative staining method. The isolated plasma-derived EVs were diluted 1:5, and the suspensions were applied to carbon-coated grids at room temperature. Carbon-coated grids were stained with 0.5% uranyl acetate and dried at room temperature in the air. The negatively stained images were captured with a 120 kV JEOL multipurpose analytical transmission electron microscope. 2.2.3 Drug loading into plasma-derived extracellular vesicles Preparation of the formulation The drug of interest, snake venom L amino acid oxidase (SV LAAO), was loaded into the plasma-derived EVs by coincubation of the SVLAAO with the EVs and freeze‒thaw cycles. Coincubation Method : For the coincubation method, 1000 µg/ml SVLAAO was coincubated with the EVs at room temperature for both 30 min and 60 min at a 1:1 ratio. The formulation was treated with an equal volume of 10% PEG and incubated at 4°C for 1 hour. The formulation was centrifuged at 10000 rpm for 1 hour at 4°C to separate the unentrapped drug. The results after 30 and 60 minutes of incubation were compared( 27 ). Freeze‒thaw cycle method : Drug loading was performed via the freeze‒thaw method by adding an SVLAAO concentration of 1000 µg/ml to equal amounts of EVs at a 1:1 ratio. The mixture was incubated at room temperature for 30 minutes, followed by freezing at -80°C for 30 minutes. Three freeze‒thaw cycles were performed to load the drug into the EVs successfully. The formulation was treated with an equal volume of 10% PEG, followed by incubation at 4°C for 1 hour. The formulation was centrifuged at 10000 rpm for 1 hour at 4°C to separate the unentrapped drug( 27 ). 2.2.3 Drug loading studies of SVLAAO-loaded extracellular vesicles: The absorbance (λmax) of SVLAAO was determined via a UV spectrophotometer. The absorbance limit was set in the range of 200–600 nm. The standard graph for SVLAAO was generated by preparing various standards of SVLAAO in the range of 1 µg/ml to 500 µg/ml. The absorbance peak of SVLAAO was determined, and the drug loading efficiency of the drug-loaded SVLAAO was determined via an indirect method by measuring the free unentrapped drug in the supernatant( 28 ). The concentration of the free drug was determined via a UV‒visible spectrophotometer at 278 nm. The encapsulation efficiency of the EVs was calculated via the following equation: 2.2.4 Drug release study of drug-encapsulated extracellular vesicles. The solutions of snake venom LAAO (2 mg/ml) and EVs containing SVLAAO were taken in dialysis bags with a 12 kDa MWCO (1 mL equivalent to 2 mg/mL) and kept in 20 mL release medium containing PBS (pH = 6.4). After the beakers containing the release medium and magnetic beads were placed on magnetic stirrers at 100 rpm, the dialysis bags containing the drugs and formulations were placed in the release medium, 800 µL of each sample was collected and replaced with the release medium every 30 minutes for 24 h, and the samples were analysed via a UV spectrophotometer at 278 nm. The concentration of each sample was calculated via a standard plot, and a release pattern graph was plotted. The data obtained from the release study were tabulated and analysed with GraphPad Prism 8.0 software. In addition to the cumulative percentage of drug release, four models of release kinetics (zero order model, first order model, Higuchi model and Korsmeyer–Peppas) were used to fit experimental data obtained from the studies. The coefficient of determination(R 2 ) values was used to select the model with better fitting for the experimental result. 2.2.5 Statistical analysis The results were analysed via GraphPad Prism version 8.0. All the data were analysed according to the Mean ± SEM. All the data were determined by paired t test and 2-way ANOVA. Values with the values P < 0.0001 were considered as significant. All experiments were conducted as n = 3. 2.3 RESULTS 2.3.1 Characterization of the Extracellular Vesicles Extracellular vesicles isolated from plasma via the polyethylene glycol precipitation (PEG) method were characterized for size via NTA, which revealed that the EVs had a mean size of 200.1 ± 66.6 nm and that the mode of the EVs was 176.1 nm (Fig. 2 ). The concentration of the particles was found to be 4.99x10 8 particles/ml. The data were analysed via NanoSight software with a detection threshold of 5. Evaluation of the ultrastructure of the isolated EVs via transmission electron microscopy (TEM) with negative staining revealed the presence of many oval or circular double-membrane structures whose average size was < 200 nm as depicted in Table 1 and Fig. 3 . Table 1 TABLE DEPICTING THE SIZE OF THE EVS DETERMINED BY THE IMAGEJ SOFTWARE. Label Area Mean StdDev Length 1 112.594 160.903 22.126 183.662 2 125.312 143.928 19.855 204.391 3 91.646 161.623 25.913 149.132 4 138.03 132.22 18.098 225.125 5 Mean 116.896 149.668 21.498 190.578 6 SD 19.778 14.219 3.374 32.403 7 Min 91.646 132.22 18.098 149.132 8 Max 138.03 161.623 25.913 225.125 Table 1 : Table describing the Mean size(in nm) of the EVs using ImageJ software. 2.3.2 Drug Loading Studies The absorbance (λmax) of SVLAAO was checked via a UV visible spectrophotometer in the range of 200–600 nm, and the maximum absorbance was found to be 278 nm. All further experiments were performed with a UV‒visible spectrophotometer using the obtained absorbance. The spectrum peak of SVLAAO is shown in Fig. 4. Entrapment efficiency (EE) of the EVs. The entrapment efficiency (EE) of SVLAAO-loaded EVs by the coincubation method at a 1000 µg/ml concentration for 30 minutes and 60 minutes. The entrapment efficiency was greater at 60 minutes than at 30 minutes. The entrapment efficiency of the formulation was 26 ± 0.060 and 58.08 ± 0.060 at 30 minutes and 60 minutes of incubation, respectively. This indicated that approximately 58% of the drug was entrapped in the plasma-derived EVs by the coincubation method for 60 minutes, and 26% of the drug was entrapped by coincubation for 30 minutes. The results after 30 minutes and 60 minutes of incubation were compared. Table 2 below depicts the entrapment efficiency of the EVs by coincubation methods at 30 minutes and 60 minutes. A graph comparing the percentage of entrapment efficiency at two different time points is shown in Fig. 5 . Table 2 TABLE DEPICTING THE ENTRAPMENT EFFICIENCY BY THE COINCUBATION METHOD Drug Entrapment Efficiency by Coincubation Method (30 mins) Sample concentration (ug/ml) Abs278 Value (graph) Dilution factor Final Value EE (%) Mean Standard deviation 1000 0.714 366.42 2 732.84 26.71 26.6807 0.060774 1000 0.714 366.42 2 732.84 26.71 1000 0.715 366.94 2 733.89 26.61 Drug Entrapment Efficiency by Coincubation Method (60 mins) Sample concentration (ug/ml) Abs278 Value (graph) Dilution factor Final Value EE (%) Mean Standard deviation 1000 0.416 209.57 2 419.15 58.08 58.15439 0.060774 1000 0.415 209.57 2 418.10 58.08 1000 0.415 209.05 2 418.10 58.18 Table 2 : Table depicting the entrapment efficiency (EE)% by the coincubation method at both 30 minutes and 60 minutes. The entrapment efficiency of the EVs loaded with SVLAAO by coincubation was compared with the entrapment efficiency of EVs loaded with SVLAAO by freeze‒thaw cycles. The results indicated that the EV-loaded coincubation method resulted in greater entrapment than did the freeze‒thaw cycles. The percentage of entrapment by the incubation method was 58.5 ± 0.060774, whereas the percentage of entrapment by the freeze‒thaw method with the same drug concentration was 55.80 ± 0.060774. The results of the entrapment efficiency according to the number of freeze‒thaw cycles are shown below in Table 3 . A comparison of the percentages of encapsulation efficiency of the two different methods is shown below in Fig. 6 . Table 3 TABLE DEPICTING THE ENTRAPMENT EFFICIENCY BY FREEZE‒THAW CYCLE Drug Entrapment Efficiency by Freeze‒thaw cycle Sample concentration (ug/ml) Abs278 Value (graph) Dilution factor Final Value EE (%) Mean Standard deviation 1000 0.438 221.15 2 442.31 55.76 55.80351 0.060774 1000 0.438 221.15 2 442.31 55.76 1000 0.437 220.63 2 441.26 55.87 Table 3 : Table depicting the entrapment efficiency (EE)% by freeze‒thaw cycle In Vitro Drug Release Study In vitro drug release studies conducted by checking the drug release pattern from the SVLAAO-loaded EVs revealed the slow and sustained release of the drug into the medium for 24 hours. 99% percent of the pure drug (nonencapsulated) was released into the medium over a period of 5.5 hours, as shown in Table 4 and Fig. 7 . The drug encapsulated into the EV by the coincubation method was released into the medium in approximately 8.5 hours, as shown in Table 5 and Fig. 8 , and the drug encapsulated by the freeze‒thaw cycles was released in 6.5 hours, as shown in Table 6 and Fig. 9 . The results revealed that the drug encapsulated by the coincubation method exhibited slow and sustained release compared with the drug released by the freeze‒thaw cycle method, as shown in Fig. 10 , thus indicating that the coincubation method is the most suitable technique for effective drug loading. The kinetic fit model was used to study the better fit data. Zero order model provided the better R value compared to first order, Higuchi and Korsmeyer–Peppas model, thus suggesting that the zero-order model fits perfectly for the following release study. The R 2 values of all the models are depicted in Table 7 . Table 4 DRUG RELEASE PROFILE OF THE PURE DRUG (SVLAAO) Time (in minutes) Percentage of the Pure drug (SVLAAO) released 0 0.069 ± 0.0001 30 0.90 ± 0.0001 60 3.75 ± 0.0001 90 8.68 ± 0.0001 120 15.69 ± 0.0001 150 24.16 ± 0.0001 180 33.47 ± 0.0001 210 43.95 ± 0.0001 240 55.41 ± 0.0001 270 69.09 ± 0.0001 300 83.61 ± 0.0001 330 99.93 ± 0.0001 Table 4 : The percentage of release of the pure drug (SVLAAO) Table 5 DRUG RELEASE PROFILE OF THE SVLAAO LOADED EXTRACELLULAR VESICLES (CO-INCUBATION) Time (Minutes) Release Percentage (Coincubation) 0 5.97 ± 0.0001 30 12.22 ± 0.0001 60 12.98 ± 0.0001 90 13.88 ± 0.0001 120 15.55 ± 0.0001 150 17.56 ± 0.0001 180 20.00 ± 0.0001 210 22.50 ± 0.0001 240 25.62 + 0.0001 270 30.00 ± 0.0001 300 35.06 ± 0.0001 330 42.70 ± 0.0001 360 50.95 ± 0.0001 390 56.02 ± 0.0001 420 61.51 ± 0.0001 450 67.55 ± 0.0001 480 79.84 ± 0.0001 510 93.11 ± 0.0001 Table 5 : Percentage of SVLAAO released from EVs loaded via the coincubation method. Table 6 DRUG RELEASE PROFILE OF THE SVLAAO LOADED EXTRACELLULAR VESICLES (FREEZE THAW) Time (in minutes) Release Percentage (Freeze‒thaw) 0 0.055 ± 0.0001 30 5.05 ± 0.0001 60 10.22 ± 0.0001 90 15.61 ± 0.0001 120 21.22 ± 0.0001 150 27 ± 0.0001 180 33.05 ± 0.0001 210 42.72 ± 0.0001 240 52.66 ± 0.0001 270 62.72 ± 0.0001 300 73.05 ± 0.0001 330 83.61 ± 0.0001 360 94.33 ± 0.0001 390 99.83 ± 0.0001 Table 6 : Percentage of SVLAAO released from EVs loaded via the freeze‒thaw method Table 7 TABLE DEPICTING THE BEST FIT KINETIC MODELS FOR RELEASE STUDY Group Zero-order model ( Q t = Q 0 + K 0 t ) First order model ( ln Q t = lnQ 0 + K 1 t ) Higuchi model (Qt = K H x t 1/2) Korsmeyer-Peppas model (Q t /Q ∞ = K k t n ) Pure drug R 2 = 0.946 R 2 = 0.457 R 2 = 0.76 R 2 = 0.946 Co-incubation R 2 = 0.916 R 2 = 0.688 R 2 = 0.75 R 2 = 0.71 Freeze thaw R 2 = 0.980 R 2 = 0.58 R 2 = 0.84 R 2 = 0.992 Table 7 : Table depicting the R 2 value of each kinetic model for drug release study </p DISCUSSION This study aimed to isolate extracellular vesicles (EVs) from human plasma and encapsulate SVLAAO by drug-loading methods involving coincubation and freeze‒thaw cycles. This study also investigated the release profile of the drug (SVLAAO) encapsulated in EVs via two different methods. In this study, EVs were isolated via the polyethylene glycol precipitation method. According to Otani et al. (2019), the extraction of extracellular vesicles with polyethylene glycol results in greater yields than the ultracentrifugation method does( 29 ). Zoia et al. (2022) isolated extracellular vesicles from the RBCs of human subjects via the polyethylene glycol precipitation method and reported that this method yielded greater amounts of EVs with sizes ranging from 100–300 nm( 30 ). In this study, SVLAAO was successfully loaded into plasma-derived EVs via coincubation and freeze‒thaw methods. The coincubation method involves loading drugs into EVs via incubation at room temperature for a specific period. The loading of drugs into EVs via the freeze‒thaw method involves repeated freeze‒thaw cycles lasting approximately 30 minutes each for three cycles. According to Shivakumar et al., 2023 coincubation method at room temperature is considered an apt method for loading, as it does not change or disrupt the membrane stability and integrity, thus maintaining the structure and properties of the EVs( 27 ). Gelibter et al. (2022) reported that exposure of EVs to freeze‒thaw cycles results in a reduction in the EV concentration and increased variability, thus reducing their therapeutic potential( 31 ). Moreover, treatment of EVs by freeze‒thaw cycles cause the loss of cargo, thus decreasing the potential to treat diseases( 32 ). The entrapment efficiency of SVLAAO was calculated via an indirect method in which the amount of unentrapped drug remaining in the supernatant at 278 nm was measured. According to Khalid et al., 2024, UV spectrophotometry is a non-destructive process that does not cause any structural damage to EVs, thus preserving the structure and functional integrity of EVs compared with other techniques ( 33 ). According to Lowe et al.,2024 techniques such as high-performance liquid chromatography (HPLC) may lead to underestimation of the loading efficiency depending upon the different types of loading methods used( 34 ). Additionally, the variability of the EV particle size may complicate the process of checking the entrapment efficiency of the particles via HPLC. The percentage of the entrapment efficiency of the EVs loaded with SVLAAO by coincubation was greater than that of those loaded by the freeze‒thaw cycle. This result coincides with the study performed by Shivakumar et al. (2023), in which the entrapment efficiency was slightly greater for EVs loaded by coincubation than for EVs loaded by freeze‒thaw cycles( 27 ). This may be explained by the fact that slight membrane damage causes decreased drug loading compared with the coincubation method. To analyse the amount of drug released over time, an in vitro drug release study was performed. Drug release from SVLAAO was fast, there was an immediate burst release, and 99% of the SVLAAO was released at 5.5 hours. The SVLAAO-loaded EVs obtained via the coincubation method exhibited slow and sustained release of the encapsulated SVLAAO. Approximately 93% of the drug was released within 8.5 hours. EVs loaded with SVLAAO via the freeze‒thaw method exhibited faster release of the drug than did those loaded via the former method. In this method, 99% of the drug was released in 6.5 hours. These results align with those of Cheng et al. (2019), who reported that drug release from exosomes loaded with cargo via the freeze‒thaw method causes rapid release of the drug into the media due to alterations in the exosomal membrane ( 35 ). Analysis of the kinetic release model for the drug release study was done to check the best fit model. The zero-order kinetic model exhibited R 2 values of 0.946 for the release of the pure drug from the dialysis membrane, while R 2 values of First order model, Higuchi model and Korsmeyer Peppas model were 0.457, 0.76 and 0.946 respectively. The R 2 values of the EVs loaded by Co incubation method and Freeze thaw cycles also exhibited the similar trend. But the pure drug and the EVs loaded by the freeze thaw cycles exhibited higher R 2 values in Korsmeyer Peppas model addition to the zero-order model, while the EVs loaded by the Co- Incubation method showed lower R 2 values for the Korsmeyer Peppas model. This can be correlated with the study by Li et al.,2021 proved that the zero-order model enables the constant release of the drug over a period, hence this model provides the better therapeutic effect and lesser side effects( 36 ). But Ortiz et al., 2021, reported the best fit model for the drug release from the NLC (nanostructured lipid carrier) was Korsmeyer Peppas model( 37 ). Meanwhile, Woolfson et al.,2006 reported that the delivery of the hydrophobic drugs like TMC120 showed the zero-order kinetic model of release, with short initial period of the burst( 38 ). These results show that drug (SVLAAO) release was slow and sustained in the case of EVs loaded by the coincubation method, thus making it an apt method for drug loading, and the formulation created by this method may act as the best therapeutic agent that can be targeted to different target sites. CONCLUSION AND FUTURE PERSPECTIVES In the current study, the potential drug-loading capacity of EVs isolated from the plasma of healthy individuals was determined. EVs isolated were characterized for specific characteristics such as morphology and size. The formulation was designed by loading SVLAAO into the EVs by two different methods (coincubation and freeze‒thaw cycles). The entrapment efficiency and the drug release study were performed with the formulation to confirm the effective loading method. Among the two loading methods, co-incubation of the EVs with SVLAAO for 60minutes, exhibited higher percentage of entrapment compared to the freeze thaw cycles. The release kinetics model of this formulation indicated that EVs loaded via the coincubation, and freezing method released the drug in a controlled manner (zero order release kinetics) thus, making it a novel and promising formulation for therapeutic purposes. In future studies, these nanovesicles can be used as carriers for various therapeutic agents and as the most effective therapeutics for various diseases, such as cancer and neurogenerative diseases. Since the stability of these nanovesicles is considered a limitation for their application, combining these naturally derived EVs with synthetic nanocarriers would produce more stable carriers with improved therapeutic capacity. The problem with off-target delivery can be overcome by using engineered EVs, wherein surface modification of EVs is performed to enhance their properties. The problem of large-scale production of EVs may be addressed by using larger bioreactors at lower costs at both the industrial and commercial levels. All these attempts may help create a bridge between the bench and the bedside, thus making extracellular vesicles successful therapeutic agent ABBREVATIONS ABBREVATIONS DEFINITION SVLAAO Snake venom L amino acid oxidase LAAO L -amino acid oxidase EV Extracellular vesicle CD9, CD37, CD63 Cluster of differentiation 9,37,63 ApoBDs Apoptotic bodies PEG Polyethylene glycol NTA Nanoparticle Tracking Analysis TEM Transmission Electron microscopy EE Entrapment Efficiency Declarations ETHICS APPROVAL AND CONSENT TO PARTICIPATE: All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000 (5). Informed consent was obtained from all patients included in the study. Approval was granted by the Institutional Ethics Committee of Kasturba Medical College and Kasturba Hospital on 04–07–2022 (number IEC:68/2022 ) . Consent for participation was obtained from healthy individuals through an informed consent form and a participation information form. CONSENT TO PUBLISH Not applicable. AVAILABILITY OF DATA AND MATERIALS The datasets used and/or analysed during the current study are available as supplementary files. COMPETING INTERESTS The authors declare that they have no competing interests. FUNDING The authors did not receive support from any organization for the submitted work. AUTHORS CONTRIBUTIONS DR: Conceptualization, methodology, data collection, analysis, and writing—original draft preparation; SMB: Supervision, project administration, review, and editing of the manuscript, final approval of the manuscript; VRB: Resources and manuscript review; KSRP: Validation, visualization, and critical revisions of the manuscript for important intellectual content, KS: Supervision, review,addressing reviewer comments and editing of the manuscript. All authors read and approved the final manuscript. ACKNOWLEDGEMENT We sincerely thank Kasturba Medical College (KMC), Manipal, and Manipal College of Pharmaceutical Sciences (MCOPS), Manipal, for providing the necessary facilities, resources, and support to conduct this study. Their invaluable assistance and infrastructure have been instrumental in successfully executing this research. No external funding was received for this study. References Nair A, Varghese BA, Gopi S, Jacob J. Smart drug delivery systems of natural products for inflammation: From fundamentals to the clinic. Inflammation and Natural Products. 2021 Jan 1;259–80. Zhao N, Ding B, Zhang Y, Klockow JL, Lau K, Chin FT, et al. Reactive oxygen species and enzyme dual-responsive biocompatible drug delivery system for targeted tumor therapy. Journal of Controlled Release. 2020 Aug 10;324:330–40. Subramanian S, Singireddy A, Krishnamoorthy K, Rajappan M. Nanosponges: A Novel Class of Drug Delivery System-Review [Internet]. Vol. 15, J Pharm Pharmaceut Sci (www.cspsCanada.org). 2012. Available from: www.cspsCanada.org van der Meel R, Sulheim E, Shi Y, Kiessling F, Mulder WJM, Lammers T. Smart cancer nanomedicine. Nat Nanotechnol. 2019 Nov 1;14(11):1007–17. Hoppstädter J, Dembek A, Linnenberger R, Dahlem C, Barghash A, Fecher-Trost C, et al. Toll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide. Front Immunol. 2019;10:1634. Mathieu M, Martin-Jaular L, Lavieu G, Théry C. Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication. Vol. 21, Nature Cell Biology. Nature Publishing Group; 2019. p. 9–17. El Andaloussi S, Mäger I, Breakefield XO, Wood MJA. Extracellular vesicles: Biology and emerging therapeutic opportunities. Vol. 12, Nature Reviews Drug Discovery. 2013. p. 347–57. Pap E, Pállinger É, Pásztói M, Falus A. Highlights of a new type of intercellular communication: Microvesicle-based information transfer. Vol. 58, Inflammation Research. 2009. p. 1–8. Schorey JS, Bhatnagar S. Exosome function: From tumor immunology to pathogen biology. Vol. 9, Traffic. 2008. p. 871–81. Simpson RJ, Jensen SS, Lim JWE. Proteomic profiling of exosomes: Current perspectives. Vol. 8, Proteomics. 2008. p. 4083–99. White IJ, Bailey LM, Aghakhani MR, Moss SE, Futter CE. EGF stimulates annexin 1-dependent inward vesiculation in a multivesicular endosome subpopulation. EMBO Journal. 2006 Jan 11;25(1):1–12. Shams SF, Mohammadipour M, Deyhim MR. Platelet-derived exosomes as the nano-carrier for miR-150 to modulate gene expression and cell cycle in the M07-e cell line. J Drug Deliv Sci Technol. 2023 Sep 1;86:104644. Semreen MH, Alniss HY, Mousa MK, El-Awady R, Khan F, Al-Rub KA. Quantitative determination of doxorubicin in the exosomes of A549/MCF-7 cancer cells and human plasma using ultra performance liquid chromatography-tandem mass spectrometry. Saudi Pharmaceutical Journal. 2018 Nov 1;26(7):1027–34. Gross JC, Chaudhary V, Bartscherer K, Boutros M. Active Wnt proteins are secreted on exosomes. Nat Cell Biol. 2012 Oct;14(10):1036–45. Litzinger DC, Buiting AMJ, van Rooijen N, Huang L. Effect of liposome size on the circulation time and intraorgan distribution of amphipathic poly(ethylene glycol)-containing liposomes. Biochimica et Biophysica Acta (BBA) - Biomembranes. 1994 Feb 23;1190(1):99–107. Zeng H, Guo S, Ren X, Wu Z, Liu S, Yao X. Current Strategies for Exosome Cargo Loading and Targeting Delivery. Vol. 12, Cells. MDPI; 2023. Gul R, Bashir H, Sarfraz M, Shaikh AJ, Bin Jardan YA, Hussain Z, et al. Human plasma derived exosomes: Impact of active and passive drug loading approaches on drug delivery. Saudi Pharmaceutical Journal. 2024 Jun 1;32(6). Costa AP, Xu X, Burgess DJ. Freeze-anneal-thaw cycling of unilamellar liposomes: Effect on encapsulation efficiency. Pharm Res. 2014 Jan;31(1):97–103. Kim MS, Haney MJ, Zhao Y, Mahajan V, Deygen I, Klyachko NL, et al. Development of exosome-encapsulated paclitaxel to overcome MDR in cancer cells. Nanomedicine. 2016 Apr 1;12(3):655–64. Luan X, Sansanaphongpricha K, Myers I, Chen H, Yuan H, Sun D. Engineering exosomes as refined biological nanoplatforms for drug delivery. Vol. 38, Acta Pharmacologica Sinica. Nature Publishing Group; 2017. p. 754–63. Xu M, Yang Q, Sun X, Wang Y. Recent Advancements in the Loading and Modification of Therapeutic Exosomes. Vol. 8, Frontiers in Bioengineering and Biotechnology. Frontiers Media S.A.; 2020. Alves de Melo Fernandes T, Rafaella Costa T, de Paula Menezes R, Arantes de Souza M, Gomes Martins CH, Junior NN, et al. Bothrops snake venom L-amino acid oxidases impair biofilm formation of clinically relevant bacteria. Toxicon. 2024 Feb 1;238. Offor BC, Piater LA. Snake venom toxins: Potential anticancer therapeutics. Vol. 44, Journal of Applied Toxicology. John Wiley and Sons Ltd; 2024. p. 666–85. Hurwitz SN, Conlon MM, Rider MA, Brownstein NC, Meckes DG. Nanoparticle analysis sheds budding insights into genetic drivers of extracellular vesicle biogenesis. J Extracell Vesicles. 2016 Jan 1;5(1). Rider MA, Hurwitz SN, Meckes DG. ExtraPEG: A polyethylene glycol-based method for enrichment of extracellular vesicles. Sci Rep. 2016 Apr 12;6. Welsh JA, Goberdhan DCI, O’Driscoll L, Buzas EI, Blenkiron C, Bussolati B, et al. Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. J Extracell Vesicles. 2024 Feb 1;13(2). Reddy SK, Ballal AR, Shailaja S, Seetharam RN, Raghu CH, Sankhe R, et al. Small extracellular vesicle-loaded bevacizumab reduces the frequency of intravitreal injection required for diabetic retinopathy. Theranostics. 2023;13(7):2241–55. Gan Q, Wang T. Chitosan nanoparticle as protein delivery carrier-Systematic examination of fabrication conditions for efficient loading and release. Colloids Surf B Biointerfaces. 2007 Sep 1;59(1):24–34. Otani K, Fujioka Y, Okada M, Yamawaki H. Optimal isolation method of small extracellular vesicles from rat plasma. Int J Mol Sci. 2019 Oct 1;20(19). Zoia M, Yesodha Subramanian B, Eriksson KK, Ravi MS, Yaghmaei S, Fellay I, et al. Validation of Effective Extracellular Vesicles Isolation Methods Adapted to Field Studies in Malaria Endemic Regions. Front Cell Dev Biol. 2022 May 16;10. Gelibter S, Marostica G, Mandelli A, Siciliani S, Podini P, Finardi A, et al. The impact of storage on extracellular vesicles: A systematic study. J Extracell Vesicles. 2022 Feb 1;11(2). Litschel T, Ganzinger KA, Movinkel T, Heymann M, Robinson T, Mutschler H, et al. Freeze-thaw cycles induce content exchange between cell-sized lipid vesicles. New J Phys. 2018 May 1;20(5). Khalid K, Ishak RB, Chowdhury ZZ. UV–Vis spectroscopy in non-destructive testing. Non-Destructive Material Characterization Methods, Elsevier. 2024;391–416. Lowe NM, Mizenko RR, Nguyen BB, Chiu KL, Arun V, Panitch A, et al. Orthogonal analysis reveals inconsistencies in cargo loading of extracellular vesicles. Journal of Extracellular Biology. 2024 Aug;3(8). Cheng Y, Zeng Q, Han Q, Xia W. Effect of pH, temperature and freezing-thawing on quantity changes and cellular uptake of exosomes. Vol. 10, Protein and Cell. Higher Education Press; 2019. p. 295–9. Li X, Li Q, Zhao C. Zero-Order Controlled Release of Water-Soluble Drugs Using a Marker Pen Platform. ACS Omega. 2021 Jun 1;6(21):13774–8. Ortiz AC, Yañez O, Salas-Huenuleo E, Morales JO. Development of a nanostructured lipid carrier (NLC) by a low-energy method, comparison of release kinetics and molecular dynamics simulation. Pharmaceutics. 2021 Apr 1;13(4). Woolfson AD, Malcolm RK, Morrow RJ, Toner CF, McCullagh SD. Intravaginal ring delivery of the reverse transcriptase inhibitor TMC 120 as an HIV microbicide. Int J Pharm. 2006 Nov 15;325(1–2):82–9. Additional Declarations No competing interests reported. Supplementary Files entrapmentefficiencyfinal.xlsx drugreleasecalculationsdfinalforall.xlsx Cite Share Download PDF Status: Published Journal Publication published 08 May, 2025 Read the published version in BMC Pharmacology and Toxicology → Version 1 posted Reviewers agreed at journal 08 Apr, 2025 Reviewers agreed at journal 08 Apr, 2025 Reviewers invited by journal 08 Apr, 2025 Submission checks completed at journal 08 Apr, 2025 First submitted to journal 05 Apr, 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. 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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-6004383","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":440246226,"identity":"cd4dc3e4-b280-4ff7-8386-997117f83d3d","order_by":0,"name":"Divya Ramesh","email":"","orcid":"","institution":"Manipal Academy of Higher Education MAHE Manipal","correspondingAuthor":false,"prefix":"","firstName":"Divya","middleName":"","lastName":"Ramesh","suffix":""},{"id":440246227,"identity":"ddae1e5f-7f38-43f2-bf2e-acfc37e6cf64","order_by":1,"name":"Shankar M Bakkannavar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYDACCQYGxgYwg/kAhMsOpHiI08KWAOEyE6+FxwAiQkiL/OzmZw9n1Nxj0J3d83XDzx0WefzNDIwP3rbh1mJw55i54YZjxQxmd85uu9l7RqJY4jADs+FcfFokEswkHwC9YXYjd9sN3jaJxIbDDGzSvHi0yM9I/yb54B9IS86zm3+BWuYfZmD/jU8Lw40cM8mNbWAtbLdBtmwA2sKMT4vBjZwyyZl9CTxmd46Z3ZZtkyg2PMzYLDnnHF6HbZPs+ZYgZ3a7+dnNt211eXLHmw9+eFOGx2FQAI+IBFg8EQ8SSFM+CkbBKBgFIwEAAGqGVDxHALPJAAAAAElFTkSuQmCC","orcid":"","institution":"Manipal Academy of Higher Education, MAHE Manipal","correspondingAuthor":true,"prefix":"","firstName":"Shankar","middleName":"M","lastName":"Bakkannavar","suffix":""},{"id":440246228,"identity":"fc8c3df3-4770-4e65-9f57-883f5f140701","order_by":2,"name":"Vinutha R Bhat","email":"","orcid":"","institution":"Manipal Academy of Higher Education MAHE Manipal","correspondingAuthor":false,"prefix":"","firstName":"Vinutha","middleName":"R","lastName":"Bhat","suffix":""},{"id":440246229,"identity":"6ce70387-4f56-4288-8085-38dfcffb29e3","order_by":3,"name":"K Sreedhara Ranganath Pai","email":"","orcid":"","institution":"Manipal Academy of Higher Education MAHE Manipal","correspondingAuthor":false,"prefix":"","firstName":"K","middleName":"Sreedhara Ranganath","lastName":"Pai","suffix":""},{"id":440246230,"identity":"31ddbf55-df00-486e-860d-33810f0d8528","order_by":4,"name":"Krishna Sharan","email":"","orcid":"","institution":"K S Hegde Medical Academy","correspondingAuthor":false,"prefix":"","firstName":"Krishna","middleName":"","lastName":"Sharan","suffix":""}],"badges":[],"createdAt":"2025-02-11 06:53:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6004383/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6004383/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40360-025-00938-8","type":"published","date":"2025-05-08T15:57:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80297580,"identity":"70a5f122-84db-4a9b-a59c-bc18a47d2813","added_by":"auto","created_at":"2025-04-10 08:44:57","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":24445,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of the methodology \u003c/strong\u003e(\u003cstrong\u003ecreated with BioRender.com)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6004383/v1/51fdeadaa07121af630425b7.jpg"},{"id":80295916,"identity":"4aae3cef-0e77-4bd0-aba3-62362fdbe9af","added_by":"auto","created_at":"2025-04-10 08:36:57","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":41754,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003edepicts the results of the nanoparticle tracking analysis of the plasma-derived EVs. The concentration and particle size are shown in the figure.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6004383/v1/879067144ad078e7196b3993.jpg"},{"id":80297581,"identity":"3641fdd9-62d8-4d78-a270-cb1378bd21b4","added_by":"auto","created_at":"2025-04-10 08:44:57","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":155801,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTransmission electron microscopy (TEM) image of the morphology of the EVs. The average size of the particles appears to be \u0026lt;200 nm. Scaling done with ImageJ Software.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6004383/v1/f98a8a569a708ea117fb4a73.jpg"},{"id":80297583,"identity":"0977ce17-ebfd-42bf-851c-b0b00c66e53b","added_by":"auto","created_at":"2025-04-10 08:44:57","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":59779,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpectrum peak pick report of SVLAAO\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6004383/v1/a022758e6617300e6d20a1e8.jpg"},{"id":80295933,"identity":"22cc8ff3-8357-4326-ae3e-67671a00ed0e","added_by":"auto","created_at":"2025-04-10 08:36:58","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":22484,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraph showing the percentage entrapment efficiency (EE %) of EVsloaded with SVLAAO by the coincubation method for 30 minutes and 60 minutes. The graphs were drawn viaGraphPad Prism 8. The graph shows that more drug was entrapped overa more extended period of incubation.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6004383/v1/32dab53148dd4183a4434a48.jpg"},{"id":80297584,"identity":"ea361774-89d6-4bb7-bbcd-67a6087b4a6e","added_by":"auto","created_at":"2025-04-10 08:44:57","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":39059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraph showing the percentage entrapment efficiency of the coincubation and freeze‒thawmethods. The graphs were drawn viaGraphPad Prism 8. The EE% of the Coincubationmethod is greater than that of the freeze‒thawcycles.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6004383/v1/594ec9f73431d7fc9e36f632.jpg"},{"id":80299258,"identity":"f4915711-48b1-4122-b200-8c0ca6b4ebfd","added_by":"auto","created_at":"2025-04-10 09:00:58","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":36471,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraph showing the percentage of the pure drug (SVLAAO) released over time. The graph shows that approximately 99% of the SVLAAO was released into the medium within 5.5 hours (330 minutes).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6004383/v1/365e74ea885531c20752efe6.jpg"},{"id":80295925,"identity":"d6bd68b4-0538-403d-902e-826c76ac1e85","added_by":"auto","created_at":"2025-04-10 08:36:57","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":46230,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraph showing the percentage of EV-loaded drug (SVLAAO) released by coincubation over time. The graph shows that approximately 93% of the SVLAAO was released into the medium within 8.5 hours (510 minutes).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6004383/v1/d8026f3a4fa41d5bef083be5.jpg"},{"id":80298347,"identity":"be7a4da6-8c95-466c-b431-617462880914","added_by":"auto","created_at":"2025-04-10 08:52:58","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":38662,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraph showing the percentage of EV-loaded drug (SVLAAO) released by freeze‒thaw cycles over time. The graph shows that approximately 99% of the SVLAAO was released into the medium within 6.5 hours (390 minutes).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6004383/v1/747af1256da971eed8906db7.jpg"},{"id":80295929,"identity":"c4ff0a70-e3fe-4a8d-99dd-b712c77c2781","added_by":"auto","created_at":"2025-04-10 08:36:58","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":38190,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vitro drug release of SVLAAO, SVLAAO-loaded EVs by coincubation and \u0026nbsp;SVLAAO-loaded EVs by freeze‒thaw cycles at pH 6.4\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6004383/v1/d78353c4d3b6823475d21e92.jpg"},{"id":82537433,"identity":"8bf7950b-e6f7-4ee7-bd83-27f14e2e32f4","added_by":"auto","created_at":"2025-05-12 16:05:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2436216,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6004383/v1/5705a3d0-d0a8-44ea-8590-c2f4491b45a6.pdf"},{"id":80295921,"identity":"56a8ea4a-4e89-4904-8733-d5f13dee769a","added_by":"auto","created_at":"2025-04-10 08:36:57","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":13666,"visible":true,"origin":"","legend":"","description":"","filename":"entrapmentefficiencyfinal.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6004383/v1/d977714aa8e7610ca3ebb26a.xlsx"},{"id":80295926,"identity":"9c03927a-353e-4149-81b1-d7caa5990204","added_by":"auto","created_at":"2025-04-10 08:36:57","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":46435,"visible":true,"origin":"","legend":"","description":"","filename":"drugreleasecalculationsdfinalforall.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6004383/v1/e6615447ba177b9ebcbe5351.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative Study on Drug Encapsulation and Release Kinetics in Extracellular Vesicles Loaded with Snake Venom L - amino acid oxidase","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eDrug delivery involves the formulation of pharmacologically active substances for desired therapeutic outcomes when these substances are administered. Hence, it has been considered a fast-emerging field in nanoscience in recent decades(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Although there has been a marked increase in the development of nano delivery tools, certain disadvantages exist, such as off-target delivery, lower efficacy, and reactions with other substances, thus making the system less effective(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Thus, there is a need to develop a novel, innovative drug delivery system that increases the efficacy of the reaction, reduces the off-target distribution and causes the controlled release of the drug.\u003c/p\u003e \u003cp\u003eIn recent years, the development of synthetic nanocarriers for drug delivery has tremendously increased. Liposomes, dendrimers, micelles, nanocapsules, etc., constitute the most common types of synthetic drug carriers (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Among these, liposomes, are considered the most versatile and are used in various nanoformulations(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Despite their numerous advantages, and its therapeutic efficiency, there are many disadvantages, such as off-target delivery, increased clearance from the reticuloendothelial system, and increased accumulation in the liver and other organs, thus limiting its availability in the target site.\u003c/p\u003e \u003cp\u003eIn addition to lipid-based carriers, other nanocarriers called extracellular vesicles (EVs) derived from cells have gained increased importance in the last few years. EVs are tiny lipid-bound particles that are important regulators of many pathophysiological reactions. Recent studies explain the similarities between liposomes and EVs as drug delivery systems and the presence of phospholipids. However, the difference lies in the specific key markers on the EVs, which aid in targeting specific areas(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEVs are classified into different types, such as ectosomes, exosomes, microvesicles, and apoptotic bodies. Although EVs are derived from different sources, they are classified according to their biogenesis pathway(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Exosomes are specific intraluminal vesicles formed during the maturation and development of multivesicular endosomes. The size of the exosomes is generally less than 200 nm. These tiny vesicles contain many surface markers, such as CD9, CD37 and CD63, which help with protein translocation and sorting(\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).Microvesicles or microparticles are EVs originating from the cell outer membrane. Apoptotic bodies (ApoBDs) are EVs released by cells undergoing programmed cell death or apoptosis.\u003c/p\u003e \u003cp\u003eEVs are present in almost all biological fluids, such as blood plasma, serum, urine, breast milk, and amniotic fluid(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). The most commonly used source for EV biosynthesis is blood plasma, which is the most stable and biocompatible with the body's physiological conditions. Plasma-derived EVs enhance the transport of drugs through biological barriers(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). For these reasons, plasma-derived EVs are considered the best vehicles for cargo transportation. In some instances, plasma-derived EVs are involved in several pathological conditions and, hence, can be used as biomarkers for various diseases(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Hence, EVs derived from cells or any biological fluid can be used as therapeutic agents by loading the necessary drugs into them and targeting the same site of interest.\u003c/p\u003e \u003cp\u003eEVs are believed to be involved in important physiological processes, such as the differentiation of cells(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The primary functions of EVs include transporting various substances to target organs. These vesicles act as vehicles to transport substances such as RNA species, DNA and various proteins(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). All these properties of EVs make them among the most efficient and versatile drug carriers.\u003c/p\u003e \u003cp\u003eThe loading of the EVs is based on the properties of the cargo to be loaded. Passive loading is the easiest method for loading drugs into EVs. This phenomenon occurs when the cells are cocultured with the drug of interest (cargo to be loaded) for a specific time. In this method, the cells incorporate the cargo and package it into the EVs during the latter formation. As this method is simple to perform, the loading efficiency is very low compared with that of active loading. Moreover, it is difficult to control the amount of drug loaded.\u003c/p\u003e \u003cp\u003eThe passive incubation method involves coincubation of the drug/cargo to be loaded with the EVs at room temperature for a specified period (usually 30 minutes to 60 minutes). Compared with the other techniques, coincubation is the most straightforward loading strategy. Passive loading occurs when the cargo concentration in the outer environment is high. Whereas Active loading occurs against the concentration gradient and results in the formation of pores in the EV membrane, thus resulting in efficient loading(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). The active method involves loading the cargo into the EV by disrupting its membrane(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). The active loading methods include electroporation, sonication, saponin treatment, freeze‒thaw cycles, etc. Compared to higher loading efficiency of electroporation and sonication, freeze-thaw cycles have intermediate loading efficiency.\u003c/p\u003e \u003cp\u003eLoading by freeze thaw cycles provide intermediate loading efficiency that involves disruption of the EV membrane by rapid freezing (-80\u0026deg;C) and thawing at room temperature (37\u0026deg;C) for a specified amount of time. This method is repeated for three continuous cycles so that the EV membrane is damaged, and the cargo is loaded successfully(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Here ,membrane damage is not permanent and occurs due to the formation of ice crystals on the membrane so that the water-soluble cargo can enter the EVs(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).Thus ,compared with passive loading techniques, active loading is complex, as it involves a risk of rapid EV membrane disruption, which may damage the functions of EVs(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe choice of loading method depends on the characteristics of the drug to be loaded, the physiological conditions under which the drug must act, and the desired therapeutic efficiency of the cargo(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). The optimum method for drug loading should provide maximum loading efficiency with less damage to the EV.\u003c/p\u003e \u003cp\u003eThis research highlights the potential of plasma-derived EVs as effective drug carriers. This study emphasized the loading of snake venom-derived L amino acid oxidase (SVLAAO) into extracellular vesicles isolated from plasma. SVLAAO has emerged as a potential therapeutic since it possesses antimicrobial and anticancer properties. LAAO from Bathrops snake venom can inhibit biofilm formation by \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e by altering the morphology of these species, resulting in antimicrobial properties(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Several other studies have shown that SVLAAO can induce cell death, thus inhibiting cancer cell metastasis(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). This property of the snake venom L amino acid oxidase makes it potentially therapeutic against cancer.\u003c/p\u003e \u003cp\u003eThis study involves loading snake venom-derived L-amino acid oxidase into plasma-derived extracellular vesicles via two different methods: coincubation and freeze‒thaw cycles. Because of its proven cytotoxic and apoptotic effects on the C6 rat glioma cell line in vitro, snake venom L-amino acid oxidase (SV-LAAO) was selected as the appropriate drug to be loaded into extracellular vesicles (EVs). This discovery, which was made possible by a pilot study, offered a strong rationale for additional research into the possible therapeutic use of SV-LAAO-loaded EVs in the treatment of glioblastoma.\u003c/p\u003e \u003cp\u003eThe outcome was evaluated via encapsulation efficiency and drug release studies. Moreover, this study evaluates the impact of two different loading techniques into the human plasma derived extracellular vesicles and was evaluated by the extent of the drug release, thus checking the efficiency of human plasma derived EVs as a potent drug carrier that will be targeted to glioblastoma multiforme (brain tumour) in vivo (Wistar rats).\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 MATERIALS\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSnake venom L amino acid oxidase was purchased from Sigma Aldrich (Cat no: A5147); human blood samples were collected from healthy volunteers after institutional ethical approval was obtained from Kasturba Hospital Manipal (IEC no: 68/2022). Institutional biosafety committee clearance products were obtained from the School of Life Sciences Manipal in August 2022, including polyethylene glycol (w/v, 6000, Sigma‒Aldrich 81260), phosphate buffer saline (pH 7.4,6.4), and dialysis membranes. A schematic representation of the methodology is depicted in Fig.\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 METHODS\u003c/h2\u003e \u003cp\u003e 2.2.1 Isolation of Extracellular Vesicles from Human Plasma: Extracellular vesicles were isolated from the plasma of healthy individuals via the polyethylene glycol precipitation method (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Informed consent was obtained from all individual participants included in the study. Five milliliters of blood was collected from healthy individuals in an EDTA-coated vacutainer, and the plasma was separated by centrifugation at 2500xg for 20 minutes. The separated plasma was centrifuged at 2500xg for 20 minutes to remove RBCs and other debris. The collected supernatant was treated with 2X 12% polyethylene glycol (PEG) and incubated overnight at 4°C. The mixture was centrifuged at 3300xg for 1 hour at 4°C to separate the pellet and the supernatant. The resulting pellet was resuspended in sterile PBS (1–3 ml), an equal amount of 2X 5% PEG was added, and the mixture was incubated at 4°C for 1 hour. In the final step, the mixture was centrifuged at 3300xg for 1 hour at 4°C, and the obtained pellet (EVs) was resuspended in 200 µl of sterile PBS (pH 7.4).\u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Characterization of Extracellular vesicles\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe plasma-derived EVs were characterized according to the MISEV 2023 guidelines(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). The concentration of the isolated EVs and their size were determined via nanoparticle tracking analysis (NTA) via a NanoSight LM10 (Malvern Instruments, UK). EV samples resuspended in PBS were vortexed and diluted 1:1000 to detect particles within the optimum detection limit. The experiments were performed three times to identify and confirm the EVs isolated from the plasma.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eMorphology of the EVs\u003c/span\u003e \u003c/p\u003e \u003cp\u003eThe structure of the EVs was determined via transmission electron microscopy (TEM). TEM was performed via the negative staining method. The isolated plasma-derived EVs were diluted 1:5, and the suspensions were applied to carbon-coated grids at room temperature. Carbon-coated grids were stained with 0.5% uranyl acetate and dried at room temperature in the air. The negatively stained images were captured with a 120 kV JEOL multipurpose analytical transmission electron microscope.\u003c/p\u003e \u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3 \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eDrug loading into plasma-derived extracellular vesicles Preparation of the formulation\u003c/span\u003e\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe drug of interest, snake venom L amino acid oxidase (SV LAAO), was loaded into the plasma-derived EVs by coincubation of the SVLAAO with the EVs and freeze‒thaw cycles.\u003c/p\u003e \u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCoincubation Method\u003c/strong\u003e:\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFor the coincubation method, 1000 µg/ml SVLAAO was coincubated with the EVs at room temperature for both 30 min and 60 min at a 1:1 ratio. The formulation was treated with an equal volume of 10% PEG and incubated at 4°C for 1 hour. The formulation was centrifuged at 10000 rpm for 1 hour at 4°C to separate the unentrapped drug. The results after 30 and 60 minutes of incubation were compared(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eFreeze‒thaw cycle method\u003c/span\u003e:\u003c/p\u003e \u003cp\u003eDrug loading was performed via the freeze‒thaw method by adding an SVLAAO concentration of 1000 µg/ml to equal amounts of EVs at a 1:1 ratio. The mixture was incubated at room temperature for 30 minutes, followed by freezing at -80°C for 30 minutes. Three freeze‒thaw cycles were performed to load the drug into the EVs successfully. The formulation was treated with an equal volume of 10% PEG, followed by incubation at 4°C for 1 hour. The formulation was centrifuged at 10000 rpm for 1 hour at 4°C to separate the unentrapped drug(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3 Drug loading studies of SVLAAO-loaded extracellular vesicles:\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe absorbance (λmax) of SVLAAO was determined via a UV spectrophotometer. The absorbance limit was set in the range of 200–600 nm. The standard graph for SVLAAO was generated by preparing various standards of SVLAAO in the range of 1 µg/ml to 500 µg/ml. The absorbance peak of SVLAAO was determined, and the drug loading efficiency of the drug-loaded SVLAAO was determined via an indirect method by measuring the free unentrapped drug in the supernatant(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). The concentration of the free drug was determined via a UV‒visible spectrophotometer at 278 nm. The encapsulation efficiency of the EVs was calculated via the following equation:\u003c/p\u003e\u003cp\u003e\u003cimg 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\" width=\"478\" height=\"76\"\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4 Drug release study of drug-encapsulated extracellular vesicles.\u003c/h2\u003e \u003cp\u003eThe solutions of snake venom LAAO (2 mg/ml) and EVs containing SVLAAO were taken in dialysis bags with a 12 kDa MWCO (1 mL equivalent to 2 mg/mL) and kept in 20 mL release medium containing PBS (pH = 6.4). After the beakers containing the release medium and magnetic beads were placed on magnetic stirrers at 100 rpm, the dialysis bags containing the drugs and formulations were placed in the release medium, 800 µL of each sample was collected and replaced with the release medium every 30 minutes for 24 h, and the samples were analysed via a UV spectrophotometer at 278 nm. The concentration of each sample was calculated via a standard plot, and a release pattern graph was plotted. The data obtained from the release study were tabulated and analysed with GraphPad Prism 8.0 software. In addition to the cumulative percentage of drug release, four models of release kinetics (zero order model, first order model, Higuchi model and Korsmeyer–Peppas) were used to fit experimental data obtained from the studies. The coefficient of determination(R\u003csup\u003e2\u003c/sup\u003e) values was used to select the model with better fitting for the experimental result.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.2.5 \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eStatistical analysis\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eThe results were analysed via GraphPad Prism version 8.0. All the data were analysed according to the Mean ± SEM. All the data were determined by paired t test and 2-way ANOVA. Values with the values P \u0026lt; 0.0001 were considered as significant. All experiments were conducted as n = 3.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.3 RESULTS\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Characterization of the Extracellular Vesicles\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eExtracellular vesicles isolated from plasma via the polyethylene glycol precipitation (PEG) method were characterized for size via NTA, which revealed that the EVs had a mean size of 200.1 ± 66.6 nm and that the mode of the EVs was 176.1 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The concentration of the particles was found to be 4.99x10\u003csup\u003e8\u003c/sup\u003e particles/ml. The data were analysed via NanoSight software with a detection threshold of 5. Evaluation of the ultrastructure of the isolated EVs via transmission electron microscopy (TEM) with negative staining revealed the presence of many oval or circular double-membrane structures whose average size was \u0026lt; 200 nm as depicted in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTABLE DEPICTING THE SIZE OF THE EVS DETERMINED BY THE IMAGEJ SOFTWARE.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLabel\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eArea\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eStdDev\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLength\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e112.594\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e160.903\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e22.126\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e183.662\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e125.312\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e143.928\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e19.855\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e204.391\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e91.646\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e161.623\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e25.913\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e149.132\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e138.03\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e132.22\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e18.098\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e225.125\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e116.896\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e149.668\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e21.498\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e190.578\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19.778\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e14.219\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.374\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e32.403\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMin\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e91.646\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e132.22\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e18.098\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e149.132\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMax\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e138.03\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e161.623\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e25.913\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e225.125\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e: \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eTable describing the Mean size(in nm) of the EVs using ImageJ software.\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Drug Loading Studies\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe absorbance (λmax) of SVLAAO was checked via a UV visible spectrophotometer in the range of 200–600 nm, and the maximum absorbance was found to be 278 nm. All further experiments were performed with a UV‒visible spectrophotometer using the obtained absorbance. The spectrum peak of SVLAAO is shown in Fig.\u0026nbsp;4.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eEntrapment efficiency (EE) of the EVs.\u003c/span\u003e \u003c/p\u003e \u003cp\u003eThe entrapment efficiency (EE) of SVLAAO-loaded EVs by the coincubation method at a 1000 µg/ml concentration for 30 minutes and 60 minutes. The entrapment efficiency was greater at 60 minutes than at 30 minutes. The entrapment efficiency of the formulation was 26 ± 0.060 and 58.08 ± 0.060 at 30 minutes and 60 minutes of incubation, respectively. This indicated that approximately 58% of the drug was entrapped in the plasma-derived EVs by the coincubation method for 60 minutes, and 26% of the drug was entrapped by coincubation for 30 minutes. The results after 30 minutes and 60 minutes of incubation were compared.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e below depicts the entrapment efficiency of the EVs by coincubation methods at 30 minutes and 60 minutes. A graph comparing the percentage of entrapment efficiency at two different time points is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTABLE DEPICTING THE ENTRAPMENT EFFICIENCY BY THE COINCUBATION METHOD\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"13\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"13\" nameend=\"c13\" namest=\"c1\"\u003e \u003cp\u003eDrug Entrapment Efficiency by Coincubation Method (30 mins)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample concentration (ug/ml)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbs278\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eValue (graph)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eDilution factor\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eFinal Value\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003eEE (%)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eStandard deviation\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.714\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e366.42\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e732.84\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e\u003cb\u003e26.71\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"2\" nameend=\"c12\" namest=\"c11\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003e26.6807\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.060774\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.714\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e366.42\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e732.84\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e\u003cb\u003e26.71\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.715\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e366.94\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e733.89\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e\u003cb\u003e26.61\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"13\" nameend=\"c13\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDrug Entrapment Efficiency by Coincubation Method (60 mins)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample concentration (ug/ml)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eAbs278\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eValue (graph)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eDilution factor\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eFinal Value\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eEE (%)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eStandard deviation\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" colname=\"c1\"\u003e \u003cp\u003e0.416\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e209.57\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e419.15\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e58.08\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"2\" nameend=\"c12\" namest=\"c11\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003e58.15439\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.060774\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.415\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e209.57\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e418.10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e58.08\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.415\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e209.05\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e418.10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e58.18\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e: \u003cb\u003eTable depicting the entrapment efficiency (EE)% by the coincubation method at both 30 minutes and 60 minutes.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe entrapment efficiency of the EVs loaded with SVLAAO by coincubation was compared with the entrapment efficiency of EVs loaded with SVLAAO by freeze‒thaw cycles. The results indicated that the EV-loaded coincubation method resulted in greater entrapment than did the freeze‒thaw cycles. The percentage of entrapment by the incubation method was 58.5 ± 0.060774, whereas the percentage of entrapment by the freeze‒thaw method with the same drug concentration was 55.80 ± 0.060774. The results of the entrapment efficiency according to the number of freeze‒thaw cycles are shown below in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. A comparison of the percentages of encapsulation efficiency of the two different methods is shown below in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTABLE DEPICTING THE ENTRAPMENT EFFICIENCY BY FREEZE‒THAW CYCLE\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e \u003cp\u003eDrug Entrapment Efficiency by Freeze‒thaw cycle\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample concentration (ug/ml)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbs278\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eValue (graph)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDilution factor\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFinal Value\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEE (%)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eStandard deviation\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.438\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e221.15\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e442.31\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e55.76\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e55.80351\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.060774\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.438\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e221.15\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e442.31\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e55.76\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.437\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e220.63\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e441.26\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e55.87\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e: \u003cb\u003eTable depicting the entrapment efficiency (EE)% by freeze‒thaw cycle\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eIn Vitro Drug Release Study\u003c/span\u003e \u003c/p\u003e \u003cp\u003eIn vitro drug release studies conducted by checking the drug release pattern from the SVLAAO-loaded EVs revealed the slow and sustained release of the drug into the medium for 24 hours. 99% percent of the pure drug (nonencapsulated) was released into the medium over a period of 5.5 hours, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003e. The drug encapsulated into the EV by the coincubation method was released into the medium in approximately 8.5 hours, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003e, and the drug encapsulated by the freeze‒thaw cycles was released in 6.5 hours, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e9\u003c/span\u003e. The results revealed that the drug encapsulated by the coincubation method exhibited slow and sustained release compared with the drug released by the freeze‒thaw cycle method, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003e, thus indicating that the coincubation method is the most suitable technique for effective drug loading. The kinetic fit model was used to study the better fit data. Zero order model provided the better R value compared to first order, Higuchi and Korsmeyer–Peppas model, thus suggesting that the zero-order model fits perfectly for the following release study. The R\u003csup\u003e2\u003c/sup\u003e values of all the models are depicted in Table \u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDRUG RELEASE PROFILE OF THE PURE DRUG (SVLAAO)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime (in minutes)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePercentage of the Pure drug (SVLAAO) released\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.069 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.90 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.75 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.68 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.69 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.16 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33.47 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43.95 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e240\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55.41 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e270\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e69.09 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e83.61 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e330\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.93 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e: \u003cb\u003eThe percentage of release of the pure drug (SVLAAO)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDRUG RELEASE PROFILE OF THE SVLAAO LOADED EXTRACELLULAR VESICLES (CO-INCUBATION)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime (Minutes)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRelease Percentage (Coincubation)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.97 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.22 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.98 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.88 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.55 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17.56 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.00 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.50 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e240\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25.62 + 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e270\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.00 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35.06 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e330\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.70 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e360\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50.95 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e390\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56.02 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e420\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e61.51 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e450\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e67.55 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e480\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e79.84 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e510\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e93.11 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e: \u003cb\u003ePercentage of SVLAAO released from EVs loaded via the coincubation method.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDRUG RELEASE PROFILE OF THE SVLAAO LOADED EXTRACELLULAR VESICLES (FREEZE THAW)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime (in minutes)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRelease Percentage (Freeze‒thaw)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.055 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.05 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.22 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.61 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.22 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33.05 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.72 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e240\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52.66 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e270\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e62.72 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73.05 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e330\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e83.61 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e360\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e94.33 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e390\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.83 ± 0.0001\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e: \u003cb\u003ePercentage of SVLAAO released from EVs loaded via the freeze‒thaw method\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTABLE DEPICTING THE BEST FIT KINETIC MODELS FOR RELEASE STUDY\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZero-order model\u003c/p\u003e \u003cp\u003e(\u003cem\u003eQ\u003c/em\u003e\u003csub\u003e\u003cem\u003et\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e= Q\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e+ K\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e\u003cem\u003et\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFirst order model\u003c/p\u003e \u003cp\u003e(\u003cem\u003eln Q\u003c/em\u003e\u003csub\u003e\u003cem\u003et\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e= lnQ\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e+ K\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e\u003cem\u003et\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHiguchi model\u003c/p\u003e \u003cp\u003e\u003cem\u003e(Qt = K\u003c/em\u003e\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e \u003cem\u003ex\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003et\u003c/em\u003e\u003csup\u003e\u003cem\u003e1/2)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKorsmeyer-Peppas model\u003c/p\u003e \u003cp\u003e\u003cem\u003e(Q\u003c/em\u003e\u003csub\u003e\u003cem\u003et\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e/Q\u003c/em\u003e\u003csub\u003e\u003cem\u003e∞\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003e= K\u003c/em\u003e\u003csub\u003e\u003cem\u003ek\u003c/em\u003e\u003c/sub\u003e\u003cem\u003et\u003c/em\u003e\u003csup\u003e\u003cem\u003en\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePure drug\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e = 0.946\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e = 0.457\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e = 0.76\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e = 0.946\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCo-incubation\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e = 0.916\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e = 0.688\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e = 0.75\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e = 0.71\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFreeze thaw\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e = 0.980\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e = 0.58\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e = 0.84\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e = 0.992\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e: \u003cb\u003eTable depicting the R\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e \u003cb\u003evalue of each kinetic model for drug release study\u003c/b\u003e\u003c/p"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study aimed to isolate extracellular vesicles (EVs) from human plasma and encapsulate SVLAAO by drug-loading methods involving coincubation and freeze‒thaw cycles. This study also investigated the release profile of the drug (SVLAAO) encapsulated in EVs via two different methods. In this study, EVs were isolated via the polyethylene glycol precipitation method. According to Otani et al. (2019), the extraction of extracellular vesicles with polyethylene glycol results in greater yields than the ultracentrifugation method does(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Zoia et al. (2022) isolated extracellular vesicles from the RBCs of human subjects via the polyethylene glycol precipitation method and reported that this method yielded greater amounts of EVs with sizes ranging from 100–300 nm(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this study, SVLAAO was successfully loaded into plasma-derived EVs via coincubation and freeze‒thaw methods. The coincubation method involves loading drugs into EVs via incubation at room temperature for a specific period. The loading of drugs into EVs via the freeze‒thaw method involves repeated freeze‒thaw cycles lasting approximately 30 minutes each for three cycles. According to Shivakumar et al., 2023 coincubation method at room temperature is considered an apt method for loading, as it does not change or disrupt the membrane stability and integrity, thus maintaining the structure and properties of the EVs(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Gelibter et al. (2022) reported that exposure of EVs to freeze‒thaw cycles results in a reduction in the EV concentration and increased variability, thus reducing their therapeutic potential(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Moreover, treatment of EVs by freeze‒thaw cycles cause the loss of cargo, thus decreasing the potential to treat diseases(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe entrapment efficiency of SVLAAO was calculated via an indirect method in which the amount of unentrapped drug remaining in the supernatant at 278 nm was measured. According to Khalid et al., 2024, UV spectrophotometry is a non-destructive process that does not cause any structural damage to EVs, thus preserving the structure and functional integrity of EVs compared with other techniques (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). According to Lowe et al.,2024 techniques such as high-performance liquid chromatography (HPLC) may lead to underestimation of the loading efficiency depending upon the different types of loading methods used(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Additionally, the variability of the EV particle size may complicate the process of checking the entrapment efficiency of the particles via HPLC. The percentage of the entrapment efficiency of the EVs loaded with SVLAAO by coincubation was greater than that of those loaded by the freeze‒thaw cycle. This result coincides with the study performed by Shivakumar et al. (2023), in which the entrapment efficiency was slightly greater for EVs loaded by coincubation than for EVs loaded by freeze‒thaw cycles(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). This may be explained by the fact that slight membrane damage causes decreased drug loading compared with the coincubation method.\u003c/p\u003e\u003cp\u003eTo analyse the amount of drug released over time, an in vitro drug release study was performed. Drug release from SVLAAO was fast, there was an immediate burst release, and 99% of the SVLAAO was released at 5.5 hours. The SVLAAO-loaded EVs obtained via the coincubation method exhibited slow and sustained release of the encapsulated SVLAAO. Approximately 93% of the drug was released within 8.5 hours. EVs loaded with SVLAAO via the freeze‒thaw method exhibited faster release of the drug than did those loaded via the former method. In this method, 99% of the drug was released in 6.5 hours. These results align with those of Cheng et al. (2019), who reported that drug release from exosomes loaded with cargo via the freeze‒thaw method causes rapid release of the drug into the media due to alterations in the exosomal membrane (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAnalysis of the kinetic release model for the drug release study was done to check the best fit model. The zero-order kinetic model exhibited R\u003csup\u003e2\u003c/sup\u003e values of 0.946 for the release of the pure drug from the dialysis membrane, while R\u003csup\u003e2\u003c/sup\u003e values of First order model, Higuchi model and Korsmeyer Peppas model were 0.457, 0.76 and 0.946 respectively. The R\u003csup\u003e2\u003c/sup\u003e values of the EVs loaded by Co incubation method and Freeze thaw cycles also exhibited the similar trend. But the pure drug and the EVs loaded by the freeze thaw cycles exhibited higher R\u003csup\u003e2\u003c/sup\u003e values in Korsmeyer Peppas model addition to the zero-order model, while the EVs loaded by the Co- Incubation method showed lower R\u003csup\u003e2\u003c/sup\u003e values for the Korsmeyer Peppas model. This can be correlated with the study by Li et al.,2021 proved that the zero-order model enables the constant release of the drug over a period, hence this model provides the better therapeutic effect and lesser side effects(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). But Ortiz et al., 2021, reported the best fit model for the drug release from the NLC (nanostructured lipid carrier) was Korsmeyer Peppas model(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Meanwhile, Woolfson et al.,2006 reported that the delivery of the hydrophobic drugs like TMC120 showed the zero-order kinetic model of release, with short initial period of the burst(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThese results show that drug (SVLAAO) release was slow and sustained in the case of EVs loaded by the coincubation method, thus making it an apt method for drug loading, and the formulation created by this method may act as the best therapeutic agent that can be targeted to different target sites.\u003c/p\u003e"},{"header":"CONCLUSION AND FUTURE PERSPECTIVES","content":"\u003cp\u003eIn the current study, the potential drug-loading capacity of EVs isolated from the plasma of healthy individuals was determined. EVs isolated were characterized for specific characteristics such as morphology and size. The formulation was designed by loading SVLAAO into the EVs by two different methods (coincubation and freeze‒thaw cycles). The entrapment efficiency and the drug release study were performed with the formulation to confirm the effective loading method. Among the two loading methods, co-incubation of the EVs with SVLAAO for 60minutes, exhibited higher percentage of entrapment compared to the freeze thaw cycles. The release kinetics model of this formulation indicated that EVs loaded via the coincubation, and freezing method released the drug in a controlled manner (zero order release kinetics) thus, making it a novel and promising formulation for therapeutic purposes.\u003c/p\u003e\u003cp\u003eIn future studies, these nanovesicles can be used as carriers for various therapeutic agents and as the most effective therapeutics for various diseases, such as cancer and neurogenerative diseases. Since the stability of these nanovesicles is considered a limitation for their application, combining these naturally derived EVs with synthetic nanocarriers would produce more stable carriers with improved therapeutic capacity. The problem with off-target delivery can be overcome by using engineered EVs, wherein surface modification of EVs is performed to enhance their properties. The problem of large-scale production of EVs may be addressed by using larger bioreactors at lower costs at both the industrial and commercial levels. All these attempts may help create a bridge between the bench and the bedside, thus making extracellular vesicles successful therapeutic agent\u003c/p\u003e"},{"header":"ABBREVATIONS","content":"\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e\u003ccolgroup cols=\"2\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eABBREVATIONS\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDEFINITION\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSVLAAO\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSnake venom L amino acid oxidase\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLAAO\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL -amino acid oxidase\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEV\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExtracellular vesicle\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD9, CD37, CD63\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCluster of differentiation 9,37,63\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eApoBDs\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eApoptotic bodies\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePEG\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePolyethylene glycol\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNTA\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNanoparticle Tracking Analysis\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTEM\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTransmission Electron microscopy\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEE\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEntrapment Efficiency\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cu\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE:\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000 (5). Informed consent was obtained from all patients included in the study. Approval was granted by the Institutional Ethics Committee of Kasturba Medical College and Kasturba Hospital on 04–07–2022 (number \u003cstrong\u003eIEC:68/2022\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e Consent for participation was obtained from healthy individuals through an informed consent form and a participation information form.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eCONSENT TO PUBLISH\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAVAILABILITY OF DATA AND MATERIALS\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available as supplementary files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eCOMPETING INTERESTS\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eFUNDING\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors did not receive support from any organization for the submitted work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAUTHORS CONTRIBUTIONS\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDR: Conceptualization, methodology, data collection, analysis, and writing—original draft preparation; SMB: Supervision, project administration, review, and editing of the manuscript, final approval of the manuscript; VRB: Resources and manuscript review; KSRP: Validation, visualization, and critical revisions of the manuscript for important intellectual content, KS: Supervision, review,addressing reviewer comments and editing of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eACKNOWLEDGEMENT\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank Kasturba Medical College (KMC), Manipal, and Manipal College of Pharmaceutical Sciences (MCOPS), Manipal, for providing the necessary facilities, resources, and support to conduct this study. Their invaluable assistance and infrastructure have been instrumental in successfully executing this research. No external funding was received for this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNair A, Varghese BA, Gopi S, Jacob J. Smart drug delivery systems of natural products for inflammation: From fundamentals to the clinic. Inflammation and Natural Products. 2021 Jan 1;259\u0026ndash;80. \u003c/li\u003e\n\u003cli\u003eZhao N, Ding B, Zhang Y, Klockow JL, Lau K, Chin FT, et al. 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Effect of pH, temperature and freezing-thawing on quantity changes and cellular uptake of exosomes. Vol. 10, Protein and Cell. Higher Education Press; 2019. p. 295\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eLi X, Li Q, Zhao C. Zero-Order Controlled Release of Water-Soluble Drugs Using a Marker Pen Platform. ACS Omega. 2021 Jun 1;6(21):13774\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eOrtiz AC, Ya\u0026ntilde;ez O, Salas-Huenuleo E, Morales JO. Development of a nanostructured lipid carrier (NLC) by a low-energy method, comparison of release kinetics and molecular dynamics simulation. Pharmaceutics. 2021 Apr 1;13(4). \u003c/li\u003e\n\u003cli\u003eWoolfson AD, Malcolm RK, Morrow RJ, Toner CF, McCullagh SD. Intravaginal ring delivery of the reverse transcriptase inhibitor TMC 120 as an HIV microbicide. Int J Pharm. 2006 Nov 15;325(1\u0026ndash;2):82\u0026ndash;9. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-pharmacology-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"phat","sideBox":"Learn more about [BMC Pharmacology and Toxicology](http://bmcpharmacoltoxicol.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/phat/Default.aspx","title":"BMC Pharmacology and Toxicology","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Extracellular vesicles, Snake venom L amino acid oxidase, Encapsulation efficiency, Drug delivery, Nanoparticles","lastPublishedDoi":"10.21203/rs.3.rs-6004383/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6004383/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThis study aimed to evaluate the potential of plasma-derived extracellular vesicles (EVs) as drug delivery carriers by employing two drug-loading techniques: coincubation and freeze‒thaw cycles.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eEVs isolated via the polyethylene glycol (PEG) precipitation method were characterized via nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The size of the particles was 200.1\u0026thinsp;\u0026plusmn;\u0026thinsp;66.6 nm. The isolated vesicles were loaded with 1000 \u0026micro;g/ml snake venom L amino acid oxidase (SVLAAO) via the coincubation method and subjected to freeze‒thaw cycles to prepare a novel formulation. The encapsulation efficiency (EE) of the loaded EVs was analysed at 30 and 60 minutes, and in vitro drug release profiles were evaluated for both methods and kinetic model for the same was determined.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe coincubation method achieved an EE of 58.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.060% after 60 minutes, which was greater than that of the freeze‒thaw method (55.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.060%). Drug release studies demonstrated that 93% of the drug was released in 8.5 hours by the coincubation method, whereas the freeze‒thaw method resulted in faster release (99% in 6.5 hours) due to membrane disruption. The best fit value (R\u003csup\u003e2\u003c/sup\u003e) was highest for zero order kinetics model.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn conclusion, the coincubation method preserves EV membrane integrity, enabling sustained drug release, making it a promising strategy for targeted drug delivery applications. This study highlights plasma-derived EVs as innovative carriers for therapeutic delivery.\u003c/p\u003e","manuscriptTitle":"Comparative Study on Drug Encapsulation and Release Kinetics in Extracellular Vesicles Loaded with Snake Venom L - amino acid oxidase","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-10 08:36:53","doi":"10.21203/rs.3.rs-6004383/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"284091715038403248543064974964178331991","date":"2025-04-08T17:50:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"269191418632056121361076963464146662237","date":"2025-04-08T12:16:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-08T11:20:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-08T06:24:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Pharmacology and Toxicology","date":"2025-04-05T05:35:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pharmacology-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"phat","sideBox":"Learn more about [BMC Pharmacology and Toxicology](http://bmcpharmacoltoxicol.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/phat/Default.aspx","title":"BMC Pharmacology and Toxicology","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"eb8ab8bd-48bd-40e4-bef7-bcb0d921785b","owner":[],"postedDate":"April 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-12T15:59:01+00:00","versionOfRecord":{"articleIdentity":"rs-6004383","link":"https://doi.org/10.1186/s40360-025-00938-8","journal":{"identity":"bmc-pharmacology-and-toxicology","isVorOnly":false,"title":"BMC Pharmacology and Toxicology"},"publishedOn":"2025-05-08 15:57:00","publishedOnDateReadable":"May 8th, 2025"},"versionCreatedAt":"2025-04-10 08:36:53","video":"","vorDoi":"10.1186/s40360-025-00938-8","vorDoiUrl":"https://doi.org/10.1186/s40360-025-00938-8","workflowStages":[]},"version":"v1","identity":"rs-6004383","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6004383","identity":"rs-6004383","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}