Preparation Irinotecan Hydrochloride Loaded PEGylated Liposomes Using Novel Method Supercritical Fluid and Condition Optimized by Box-Behnken Design

Preparation Irinotecan Hydrochloride Loaded PEGylated Liposomes Using Novel Method Supercritical Fluid and Condition Optimized by Box-Behnken Design | 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 Preparation Irinotecan Hydrochloride Loaded PEGylated Liposomes Using Novel Method Supercritical Fluid and Condition Optimized by Box-Behnken Design Misagh Mohammadi, Mehrnaz Karimi, Farhad Raofie This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4492900/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Sep, 2024 Read the published version in Discover Nano → Version 1 posted 9 You are reading this latest preprint version Abstract A semi-synthetic camptothecin derivative known as irinotecan hydrochloride is frequently used to treat colorectal cancer (CRC), including colorectal adenocarcinoma and lung cancers involving small cells. Irinotecan has a very short half-life; therefore, continuous infusions are required to keep the drug's blood levels at therapeutic levels, which could produce cumulative toxicities. Effective delivery techniques, including liposomes, have been developed to address these shortcomings. In this study, a continuous supercritical fluid approach dubbed Expansion Supercritical Fluid into an aqueous solution (ESSAS), in which the pressure decreases rapidly but remains over the critical pressure, is proposed to manufacture polyethylene glycolylated (PEGylated) liposomes carrying irinotecan hydrochloride. To accomplish this, PEGylated liposomes were created using a Box-Behnken design, and the operating parameters (flow rate, temperature, and pressure drop) were optimized. Encapsulation efficiency, mean size, and prepared liposome count were 94.6%, 55 nm, and 758 under ideal circumstances. Additionally, the stability of the PEGylated liposome was investigated during 8 weeks, and also PEGylated liposome-loaded irinotecan release profile was compared to conventional liposomes and free irinotecan, and a constant drug release was seen after the first burst release from liposomes. Drug delivery systems Supercritical fluid expansion Liposomes Irinotecan Polyethylene glycol Response surface methodolog Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Nanotechnology has recently begun to develop in various fields, including cosmetics, pharmaceuticals, medicine, food, and environmental research. Most pharmaceutical research has focused on drug delivery systems (DDS). Developing nanocarriers, such as lipid carriers (known as liposomal formulations), is one of the most promising strategies for drug delivery systems, as they significantly improve drug pharmacokinetics and, in turn, reduce toxicity and improve therapeutic efficacy and biocompatibility of drugs [ 1 , 2 ]. These spherical vesicles are composed of one or more phospholipid bilayers. These carriers are biocompatible because they resemble human cells whose exterior lipid bilayer encloses the aqueous compartment [ 3 , 4 ]. Moreover, liposomes are advantageous drug carriers because they can constantly release the captured substrates, which include both hydrophilic and hydrophobic substrates [ 5 ]. Liposomes can also provide a slow drug release, resulting in long-term drug exposure to tumor cells and improving their efficacy[ 6 ]. The mononuclear phagocyte system (MPS), which ingests and eliminates liposomes circulating in the bloodstream, is assumed to be the mechanism by which liposomes are cleared from the body. So, it is especially crucial to prevent capture by the liver's and spleen's phagocytic cells to prolong blood circulation time. Chain length, lipid unsaturation, lipid composition, size, and Zeta potential are just a few of the liposome properties that impact how long they circulate through the blood [ 7 – 9 ]. Recent research has shown that some liposome formulations, such as those with a small amount of phospholipid modified by polyethylene glycol (PEG), allow these carriers to remain in the blood for longer periods [ 5 , 10 , 11 ]. Although the specific mechanism of the MPS avoidance phenomenon is unknown, it is thought that the PEG chains, due to their water-binding ability and flexibility, prevent the uptake of opsonizing proteins that guide liposomes to macrophages [ 12 ]. Furthermore, PEG can protect liposomes by producing a polymer layer on the liposome surface, increasing the hydrophilicity of the surface, increasing the mutual repulsion between the polymer coating and blood components, and shielding surface charge [ 13 ]. Also, through an increased permeability and retention (EPR) effect via a passive targeting mechanism, liposomes modified with polyethylene glycol spontaneously accumulate in solid tumors [ 1 ]. PEGylated liposomes offer a desirable platform for enhancing the therapeutic index of a range of anticancer medications [ 5 ]. Irinotecan hydrochloride (IRH), a semisynthetic derivative of camptothecin, is frequently used in treating colorectal cancer (CRC) such as colorectal adenocarcinoma and malignancies related to small cells in the lung [ 14 , 15 ]. IRH has been found to specifically engage with the topoisomerase, which controls DNA topology and carries out several nuclear functions, including DNA replication, recombination, and repair. So, it can prevent DNA replication in tumor cells in the case of CRC [ 16 ]. Currently, the use of this substance as a single agent or in combination with other chemotherapeutic drugs is approved for treating colorectal, ovarian, cervical, and small-cell lung cancer [ 17 ]. But it has been found that IRH has dosage-dependent side effects such as myelosuppression and severe diarrhea because of the bis-piperidine group, which is known to constitute dose-limiting toxicity and restrict the dose that can be used safely [ 14 , 18 ]. Furthermore, due to the extremely short half-life of IRH, continuous infusions are necessary to maintain its therapeutically effective blood level, which can lead to cumulative toxicities. Because of these drawbacks, effective delivery strategies can both (a) avoid the hydrolytic conversion of the lactone configuration of the E-ring to an inactive open-ring carboxylate and (b) provide prolonged IRH release have been developed [ 17 ]. One of the most efficient drug delivery strategies is the liposomal formulation. Current studies claim that IRH is favorable to effective encapsulation in pharmaceutically viable liposome systems due to its physicochemical properties. These liposomal formulations can decrease IRH in vivo clearance and boost anticancer effectiveness [ 14 ]. In 2015, the irinotecan liposome injectable Onivyde received US FDA (Food and Drug Administration) approval to treat patients with advanced pancreatic cancer [ 17 ]. Liposomes can be made using several methods, with at least 14 major methods reported. Lipid film hydration, also known as thin-layer evaporation, rehydration-dehydration, reverse-phase evaporation, ethanol injection, French press, ether infusion, and detergent dialysis techniques, are the seven most widely used methods [ 19 ]. These techniques have various drawbacks, such as difficult control of particle size distribution, low encapsulation efficiency, high liposome size, and low method reproducibility due to discontinuous layouts [ 20 ]. Moreover, the methods above require a large value of organic solvents, which are harmful to the environment and the human body. Due to the need to completely remove any leftover organic solvent, the use of organic solvents in manufacturing drug delivery carriers must be avoided or minimized as much as feasible [ 19 ]. Although there is a way to create liposomes by repeatedly freezing and melting an aqueous solution, it is evident that this method requires a lot of energy. So, these techniques are unsuitable for producing liposomes in large quantities because they involve numerous steps and cannot fully overcome those limitations [ 3 , 21 ]. Supercritical fluid (SCF) has been considered in producing nanoliposomes to reduce or eliminate the use of organic solvents and achieve greener chemistry [ 22 ]. When a fluid is in the supercritical state, it combines the physicochemical characteristics of both liquids and gases, including high density and viscosity. These characteristics are adjustable with changes in pressure and temperature [ 19 , 23 ]. The most widely used SCF is carbon dioxide because it is cheap, nontoxic, and has a low critical temperature (31.1°C) and pressure (7.38 MPa), making it safe and suitable for handling pharmaceutical chemicals. Moreover, after depressurization, CO 2 reverts to its gaseous state, It prevents any problem caused by the toxicity of residual solvents in the final product [ 21 ]. In contrast to conventional ones, these processes are quick,easy, and create liposomes with nanometric dimensions; as a result, the need for post-processing steps like extrusion or sonication is frequently dropped. Moreover, liposomes are produced with greater physicochemical property control and reproducibility, making these methods suitable for industrial production under GMP (Good manufacturing practice) guidelines. In addition, due to the properties of SCF, the use of organic solvents can be controlled or eliminated [ 6 , 24 ]. SCF can be used as an antisolvent in the SAS (Supercritical antisolvent), ASES (Aerosol solvent extraction system), or GAS (Gas Antisolvent) processes, in which an organic solvent is utilized to solubilize lipids, which are subsequently precipitated by the antisolvent effect of SCF. Moreover, The SCF could be used as a dispersing agent. These techniques involve dispersing the lipids in pure water, followed by applying SCF pressure to cause fine dispersion of the lipids due to collision and shear forces [ 21 , 25 , 26 ]. In this procedure, the phospholipids are evenly diffused into the medium, and liposomes are generated during depressurization. Moreover, another option is that SCF operates as a lipid solvent, such as Rapid Expansion of Supercritical Solution (RESS), supercritical Assisted liposome formation (SuperLip), and Expansion Supercritical Fluid into an aqueous solution (ESSAS) [ 27 – 30 ]. In general, the effectiveness of the supercritical fluid's ability to generate nano-liposomes depends on several factors, including flow rate, pressure, time, temperature, etc. In the circumstances like this, when many factors may affect the final result, optimizing and modeling process variables using approaches such as the one factor at a time (OFAT) or design of experiments (DOE) approach is an excellent way to increase process efficiency [ 31 ]. Since in the DOE strategy, all the interactions between various parameters are considered, it produces more accurate findings than the OFAT strategy. In addition, DOE requires fewer experiments to achieve ideal conditions, so this method is more valuable than the single-factor method [ 32 , 33 ]. One of the DOEs is the Behnken box (BB) design, created by combining an incompletely balanced or semi-balanced block design with a two-level factorial design in a special way and enables the rapid estimation of first- and second-order coefficients of the mathematical mode [ 34 ]. Furthermore, the three-component Box-Behnken matrix has a spherical, rotatable design, which means that all design points (aside from the center) are dispersed uniformly across the sphere [ 35 ]. This research aims to develop irinotecan hydrochloride PEG-coated liposomes using the ESSAS method previously introduced by our team [ 28 , 36 ]. ESSAS is a modified version of RESS and supercritical fluid extraction of emulsions [ 37 , 38 ] in which the pressure suddenly drops, but it remains higher than the critical pressure. As a result, phospholipids' precipitation rate can be controlled, and homogeneous liposomes with narrow size distribution can be produced. Furthermore, whereas in other processes, the pressure is brought down to atmospheric pressure from above the critical pressure, this procedure allows for producing massive liposomes with a depressurization rate of fewer than 10 MPa. Also, we employ BB to research and optimize the variables influencing nanoliposome size, encapsulation efficiency, the number of nanoliposomes formed, and vesicle stability. Measurements will be made using the Zeta potential and the Field Emission Electron Microscope (FESEM) to show the stability, size, and morphology of the generated liposomes. To confirm the PEG-coating's success and manage drug release kinetics, the release of irinotecan hydrochloride will be investigated. 2. Experimental 2.1.Materials Cholesterol, m-dPEG® 12 -amido-dPEG® 24 -DSPE (mPEG 1500 -DSPE), and 1,2-Distearoyl-sn-glycerol-3- phosphocholine (DSPC) were purchased from Sigma-Aldrich. Carbon dioxide with a purity of more than 99.9% was supplied by Roham Co. in Tehran, Iran. Moreover, Aventis Pharma ( Mumbai , India) provided the Irinotecan hydrochloride standard used to encapsulate. HPLC-grade ethanol and methanol were obtained from Caledon (Georgetown, Ont., Canada). Furthermore, all formulations were created using distilled and deionized water. 2.2.Preparation of Nanoliposomes 2.2.1. Preparation of the phospholipid cargo solution At 25°C, 120 mg DSPC and 30 mg mPEG1500-DSPE were dissolved in a 40 mL mixture of ethanol/water (30% V/V), and the desired solution was then supplemented with 25 mg cholesterol and 30 mg irinotecan hydrochloride standard. After that, the solution of the active ingredients was then stirred for 90 minutes at 1100 rpm and afterward, 2 mL of the resulting solution was transferred to the equilibrium vessel for liposome preparation. 2.2.2. Nanoliposome production process In this investigation, the expansion Supercritical Fluid into an aqueous solution (ESSAS) approach, was utilized to create liposomes on a Suprex automated Prepmaster 44 system (AP44) with a variable flow restrictor (Pittsburgh, PA). Figure 1 depicts a schematic of the ESSAS apparatus used to create liposomes. Three valves and two high-pressure vessels, referred to as the equilibration and production vessels, with inner volumes of 10 cm 3 and dimensions of 16 mm i.d and 141 mm height, formed the device's overall layout. Also, the vessels' temperatures were tracked using a thermocouple housed inside the oven. A digital pressure gauge was used to assess the pressure inside the high-pressure vessels. To create PEGylated liposomes, 2 mL of the cargo solution was first moved to the equilibration vessel, and 7 mL of HPLC-grade water was added to the production vessel. To achieve the highest EE, the greatest number of produced liposomes, and the smallest size of vesicles, the parameters of temperature, flow rate, and pressure drop (Pd), whose range were determined by the initial tests, were studied using Statgraphics XVII software and Box-Behnken designs. Furthermore, the collection time, equilibrium pressure, and equilibrium time were held constant for all experiment runs at 15 minutes, 40 MPa, and 30 minutes, respectively. Because the solubility of supercritical fluid increases with increasing pressure, and the equilibrium pressure was set at the highest operating pressure of the device at 40 MPa. Also, after 15 minutes, all the solution in vessel1 was transferred to vessel 2, and higher collection times were practically useless. In the first stage, by opening valve 1, the condensed carbon dioxide produced by the high-pressure pump was transferred to the equilibrium vessel through a capillary tube. The cargo solution was allowed to dissolve in carbon dioxide for 30 minutes once the pressure hit 40 MPa and the temperature reached the value determined by the software. After the equilibrium time was up, valves 2 and 3 were opened to create the pressure drop, and the contents of the equilibrium vessel were expanded into the production vessel while supercritical CO 2 (SC-CO 2 ) was passed from it. At this stage, a sudden pressure drop occurred. As a result, the density and solubility of SC-CO 2 decreased, and using nucleation, substances dissolved in SC-CO 2 began to precipitate in the production vessel. Eventually, during this procedure, the precipitated phospholipids, mPEG 1500 -DSPE, and cholesterol were brought together and spontaneously produced nanoliposomes containing a portion of IRH. The production vessel achieved ambient pressure when the operation was finished because valve 3 was left open while valves 1 and 2 were closed. The resultant solution was then collected in a vial and kept at 4 ° C for future analysis. 2.3.Characterization of Liposomes: morphology and size distribution 2.3.1. Field Emission Scanning Electron Microscopy (FESEM) A field emission scanning electron microscope was used to investigate the size and morphology of liposomes (FESEM, MIRA3 TESCAN-XMU, and the Czech Republic). In specifics, two droplets of the nanoliposome suspension were sprayed on the aluminum bases and allowed to air dry. Then, a layer of platinum was applied to dry liposome suspension using a sputtering apparatus (Pelco SC-7, Ted Pella Inc., Redding, CA). The liposomes were subsequently seen using an accelerating voltage of up to 10 kV at a magnification of 250 and 90,000 times. The images were analyzed using the ImageJ software. 2.3.2. Size distribution, and Zeta potential of liposomes Following the ESSAS process, Dynamic Light Scattering (mod. Zetasizer Nano S, United Kingdom) was used to measure the mean diameter (MD) and zeta potential (ZP) of the generated nanoliposomes on each sample. This device has a 633 nm wavelength, 5.0 mW He-Ne laser with a 173-degree scattering angle. Each test required 1 mL of the nanoliposome suspension, and measurements were made at room temperature in triplicates. 2.4.Storage stability study The storage stability of the nanoliposome suspension produced by the ESSAS method was examined for up to 8 weeks. At specific time intervals, the nanoliposome's EE and size were examined for storage stability. 2.5.Determination of Encapsulation Efficiency (EE) The encapsulation efficiency of the manufactured PEGylated liposomes was investigated by measuring non-encapsulated IRH in the generated liposome suspension. We determined the amount of irinotecan hydrochloride entrapped in the nanoliposomes by comparing the initial amount of the drug used to make the cargo solution to the quantity of unencapsulated drug that remained in the liposome suspension after production. To do this, the liposome suspension created using the ESSAS approach was centrifuged at 4600 rpm for 75 minutes. After that, the supernatants were separated, and irinotecan hydrochloride concentrations were determined using high-performance liquid chromatography (HPLC). A liquid chromatographer (HP Agilent 1050, Agilent, Santa Clara, California, USA) in conjunction with a variable wavelength detector and HP ChemStation software was used to carry out the HPLC method for determining and monitoring the irinotecan hydrochloride content in supernatants. The separation was performed using an Agilent HT Zorbax SB-C18 column (5 µm particle size, 4.6 mm 250 mm; Agilent Technologies, Santa Clara, CA) with 10 µL of sample injection volume. The mobile phase in the isocratic elution protocol was 3% V/V triethylammonium acetate (TEAA) buffer (pH 3) and acetonitrile (70:30 V/V). The mobile phase flow rate and detector wavelength were constant throughout the 25-minute analysis at 1 mL/min and 379 nm, respectively. Irinotecan hydrochloride standard curves were created using the drug standard at 10 to 500 ppm concentrations before analyzing the supernatants. The encapsulation efficiency is then assessed using Eq. 1, where CT denotes the initial amount of IRH and F denotes the amount of free irinotecan hydrochloride in the liposome suspension. \(\text{E}\text{E}\text{\%} =\frac{\text{C}\text{t} - \text{F}}{\text{C}\text{t} } \times 100\text{\%}\) Eq. (1) 2.6.Drug release test Drug release tests were conducted at 37 ° C using the HPLC procedure used to quantify the EE. For this purpose, A dialysis sack (MWCO 3500 Da, Sigma Aldrich, Milan, Italy) was filled with 4 mL of the generated IRH PEGylated liposomes and clamped on both sides. After that, it was placed in a 150 mL volume of distilled water and stirred at 300 rpm without any light. 1 mL aliquots of the dialysate (a distilled water bath solution containing the released IRH) were taken at fixed time intervals to calculate the quantity of drugs released and to indicate drug release profiles. Following the evaporation of the dialysate's solvent, the remaining irinotecan hydrochloride was dissolved in 200 µL of ethanol to raise the medication's concentration, and by the HPLC method amount of IRH was determined. The PEGylated liposomes that were produced under ideal circumstances were used for these studies. For comparison purposes, another release test was conducted using non-PEGylated vesicles (which were produced under the same conditions but without the presence of mPEG1500-DSPE) and pure irinotecan hydrochloride solution (10 mg drug in 10 mL water/ethanol 95% V/V). 3. Results and Discussion 3.1.Response Surface Methodology 3.1.1 Fitting the Model and Data Analysis The main objective of producing PEGylated liposomes using the ESSAS method is to speed up the preparation and manufacturing processes while improving repeatability, encapsulation effectiveness, homogeneity, liposome count, and steady release of irinotecan for a more extended time compared to liposomes which is made by conventional methods. During the equilibrium time, the cargo solution compounds in vessel 1 were dissolved and saturated in the SC-CO 2 . The pressure was then quickly reduced during the production step by opening valves 2 and 3, allowing SC-CO 2 to flow into vessel 2. This caused the creation of fine water droplets and reduced the density and solvent power of SC-CO 2 . As a result, phospholipid, cholesterol, and mPEG 1500 -DSPE were precipitated from SC-CO 2 and quickly covered water droplets that had dissolved a portion of the irinotecan in themselves. This method led to the formation of reverse lipid micelles, which then fell into the aqueous bulk of vessel 2 and were stabilized by the second layer of phospholipids and mPEG 1500 -DSPE which were covering these structures, and finally, PEGylated Irinotecan liposomes were created. Nanoliposome characteristics, including EE, the number of liposomes produced at 1 µm scale, and liposome size, are influenced by temperature, flow rate, and pressure drop. Hence the parameters were optimized using the Box Behnken design, one of the experimental designs. According to the equation K = 2K(K-1) + C, where k is the number of parameters, and C is the number of center point repetitions, 15 tests were finished with 3 center point repetitions. Also, the preliminary tests found that after 15 minutes, all of the cargo solution in vessel 1 is transferred into the production vessel. So, in all tests, the dynamic time was set at 15 minutes. Additionally, the equilibrium pressure was taken to be 40 MPa because the density of the supercritical fluid and, consequently, the solubility of the cargo solution compounds in it, increases with the increase of the equilibrium pressure. Table S1 displays the three factors with lower and higher design points in coded values. In addition, Table 1 displays the experimental conditions and the obtained outcomes. Analysis of responses seen throughout all runs was conducted using stat graphics software. It was discovered that a quadratic model provided the best fit. Additionally, the least squares method was used to compute the variance of this design and the model equation's coefficients. The results of ANOVA analysis of the experimental data for mean size, count, and EE are reported in Tables S2, S3, and S4, respectively. The significance of various coefficients, including linearity, quadraticity, and interaction, is listed in these tables along with the p-value, where the expressions of the model with p 0.05 denote significant and insignificant results, respectively. Table 1 Design matrix and the responses (BBD) for the production of irinotecan PEGylated nanoliposomes. Experiment Flow rate (mL/min) Pressure drop (MPa) Temperature (˚C) Mean size (nm) Count EE (%) Figures Run 1 2.0 22.5 52.5 53 368 86 Figure 3a Run 2 3.0 30.0 52.5 41 429 96 Figure 3c Run 3 1.0 22.5 65.0 57 170 67 Figure S3a Run 4 2.0 22.5 52.5 51 357 88 Figure S3b Run 5 3.0 22.5 65.0 74 64 81 Figure 3g Run 6 2.0 30.0 65.0 48 300 77 Figure S3c Run 7 2.0 22.5 52.5 49 350 87 Figure S3d Run 8 2.0 15.0 65.0 94 110 75 Figure S3e Run 9 3.0 22.5 40.0 72 265 89 Figure S3f Run 10 2.0 30.0 40.0 67 710 85 Figure S3g Run 11 2.0 15.0 40.0 108 60 76 Figure 3e Run 12 1.0 30.0 52.5 46 460 71 Figure S3h Run 13 1.0 22.5 40.0 75 180 76 Figure 3i Run 14 3.0 15.0 52.5 82 86 77 Figure 3l Run 15 1.0 15.0 52.5 98 60 75 Figure S3i The formulas of the second-order polynomial equations resulting from the experimental designs were obtained as follows: Y 1 = 588.895–42.125X 1 − 13.2333X 2 − 11.406X 3 + 3.0X 1 2 + 0.366667X 1 X 2 + 0.4X 1 X 3 + 0.226667X 2 2 − 0.0133333X 2 X 3 + 0.0992X 3 2 Eq. (2) Y 2 = -3542.36 + 689.717 X 1 + 84.3167 X 2 + 80.714 X 3 − 112.417 X 1 2 − 1.9 X 2 − 3.82 X 1 X 3 + 0.228148 X 2 2 − 1.22667 X 2 X 3 − 0.487467 X 3 2 Eq. (3) Y 3 = -45.58 + 2.95 X 1 + 2.78 X 2 + 3.564 X 3 − 3.625 X 1 2 + 0.766667 X 1 X 2 + 0.02 X 1 X 3 − 0.0644444 X 2 2 − 0.0186667 X 2 X 3 − 0.0328 X 3 2 Eq. (4) Where Y 1 , Y 2 , and Y 3 stand for the mean size, count of PEGylated liposomes, and EE. Furthermore, the X 1 through X 3 stands for the flow rate (mL/min), pressure drop (MPa), and temperature (°C), respectively. The ANOVA results showed good R 2 and R 2 adjusted for mean size (97% and 91%), EE (98% and 96%), and the count of produced PEGylated irinotecan liposomes (99% and 97%), respectively. The high-adjusted R-square value demonstrates a strong correlation between the estimated and measured values, which verifies the accuracy of the data and the model. The "Lack of Fit" p-values for mean size, count, and EE were 0.0589, 0.0590, and 0.2544, respectively. As is clear, they are all more than 0.05, indicating that the Lack of Fit was not significant compared to the pure error and the model correctly predicted the experimental data. Additionally, Fig. S1 displayed how well the experimental size, count, and EE values fit the expected values. These outcomes demonstrate that the model created in this study can correctly project the experimental data. It should be noted that the zeta potential is used to measure the stability of liposomes in suspension; values higher than |30| mV indicate liposome stability. The PEGylated-liposomes generated exhibited good stability in this investigation, and the ZP was always greater than − 30 mV. 3.1.2. Pressure drop effect The effect of each parameter on the characteristics of IRH PEGylated liposomes is shown using main effect graphs (Fig. 2 d, 2 e, and 2 f) when one parameter is altered while the others are kept constant. additionally, the Pareto chart (Fig. 2 a, 2 b, and 2 c) shows the parameters' impact and specifies which factors significantly impacted the responses at a 95% confidence level. Figure 2 d shows that increasing the pressure drop up to 28 MPa caused the IRH liposome size to fall from 86 to 41 nm before remaining constant. To explain this phenomenon, it can be said that a greater pressure drop caused a higher decrease in the density and solubility of SC-CO 2[19] . As a result, it created a greater difference between the value of phospholipids that could be dissolved in SC-CO 2 before and after the expansion step, leading to higher supersaturation. According to the nucleation theory, more supersaturation encouraged nucleation during the expansion step, leading to smaller liposomes. Additionally, more nucleation caused the generation of more nuclei, so as shown in Fig. 2 e, the count of produced liposomes increased. As indicated in Fig. 2 f, the encapsulation efficiency of the created liposomes increased to 87.5% until the pressure drop reached at 27 MPa and decreased thereafter. Because as previously stated, increasing the pressure drop resulted in the production of more liposomes, which increased encapsulation efficiency. However, increasing the pressure drop resulted in the production of smaller liposomes with less internal space to trap IRH. As a result, the EE decreased due to the dominance of this effect on the number of liposomes after the pressure drop of 27 MPa. Figure 3(a, b, c, and d) show the FESEM picture and particle size distribution of experiments 1 and 2, indicating the effect of pressure drop on nanoliposome production. Furthermore, these figures demonstrate that spherical liposomes with a narrow size distribution were generated in this study. Moreover, the Pareto chart (Fig. 2 a, 2 b, and 2 c) demonstrates that pressure drop significantly impacts the mean size, count, and EE of the production process. 3.1.3. Temperature effect This study looked into the effects of temperatures from 40 to 65°C. According to Fig. 2 a, 2 b, and 2 c the mean size, count, and EE of liposomes were significantly influenced by temperature. According to Fig. 2 d, 2 e, and 2 f, the temperature had a reverse effect in this study. Firstly, the rising temperature caused more phospholipid soluble in CO 2 due to the increased mass transfer speed, leading to greater supersaturation [ 39 ]. Therefore, the size of produced liposomes was reduced to 50 nm. Moreover, As the temperature rose, the energy of the molecules in the phospholipid bilayer increased. It improved the membrane's fluidity, which would encourage the generation of liposomes, make it easier for the drug to be wrapped around the wall material, and increase the amount of drug that can be loaded into each vesicle [ 40 ]. So, the count and EE of liposomes rose to 370 and 87.5%, respectively. On the other hand, as the temperature increased, the density of SC-CO 2 decreased. As a result, it decreased the solubility of the compounds and the solvent capacity of SC-CO 2 , which led to lower supersaturation and the production of bigger (61 nm) and fewer liposomes (210) with lower EE (79%). The FESEM image and particle size distribution from experiments 5 and 11 are shown in Fig. 3e, 3f, 3g, and 3h, respectively. These results demonstrate how temperature affects the production of IRH PEGylated liposomes. These images' narrow and homogeneous particle size distribution suggested that uniform PEGylated liposomes were formed. 3.1.4. Flow rate effect Figure 2 shows that the flow rate in the 1 to 3 mL/min range did not significantly affect the mean size and number of liposomes. But the EE was highly influenced by this parameter. This was expected because pressure and temperature have a greater impact on SC-CO 2 properties such as density, mass transfer, and solubility than flow rate, which therefore have a greater effect on the size and number of liposomes produced. As shown in Fig. 2 d, 2 e, and 2 f, increasing the fluid flow rate resulted in higher mixing energy and greater atomization of cargo solution droplets in SC-CO 2 flow[ 29 ]. These conditions caused liposomes to develop more quickly and around smaller droplets, leading to an increase in the number of liposomes and EE. On the other hand, after the flow rate reached 2.7 mL/min, the turbulence induced by the flow rate resulted in non-uniformity and the creation of larger liposomes, which also had larger internal space. As a result, the encapsulation efficiency remained unchanged. Figures 3i, 3j, 3k, and 3l, display the FESEM image and the particle size distribution of experiments 13 and 14, respectively. These figures show how flow rate influences the morphology, count, and homogeneity of IRH liposomes. 3.1.5. Contour plots and Response Surface analysis As functions of pressure drop, temperature, and flow rate, the response surface diagrams and contour plots of size, count, and EE of PEGylated liposomes were achieved by adjusting the two of them while the other one held constant, which is shown in Fig. 4 and S2, respectively. The response surfaces' steepness demonstrated how variables affected responses. More impact of variables on response was indicated by the steeper surfaces. Moreover, the density of contour lines displayed how various factors affected responses. The peak value in Fig. 4 (d, e, f, g, h, and i) represented the perfect value for nanoliposome count and EE. In contrast, the valley value in Fig. 4 (a, b, and c) represented the ideal value for the size of produced liposomes. The outline's shape offers additional proof of how two factors interacted with one another. Furthermore, the contours' shape (Fig. S2) also demonstrated the strong and weak interactions between the two factors. The close circle showed that there was no significant interaction between the two factors , Fig. 4 : Estimated response surface for (a,d,g) pressure drop was fixed at 22.5 MPa, (b,f,h) flow rate was fixed at 2 mL/min, (c,e,i) temperature was fixed at 52.5 ˚C for the mean size, Count, and EE, respectively. 3.1.6. Optimization condition The resulting model was used to forecast the ideal conditions for producing irinotecan PEGylated liposomes by the ESSAS method. The ideal conditions for the produced nanoliposomes included a pressure drop at 29.9 MPa, a flow rate at 2.4 mL/min, and a temperature oat 42°C to maximize the EE and count of created liposomes while minimizing the size. The predicted mean size, count, and EE of the produced liposomes could reach 58 nm, 663, and 91.5% under optimization conditions. The test was run under the aforementioned circumstances three times to evaluate the anticipated model's adaptability and optimal points with an experimental test. As seen in Fig. 5a, 758 nanoliposomes with an average size of 55 nm were created. Figure 5b also shows the size distribution of liposomes produced at the optimal point. The EE for produced nanoliposomes also reached 94.6% in this test, confirming the remarkable agreement between the projected model and the experimental test under ideal conditions. Compared to other studies illustrated in Table 2 , it is obvious that the liposomes created in this work had encapsulation efficiency equal to or greater than studies that produced irinotecan liposomes. In contrast, the size of the liposomes generated in this work was significantly smaller than those. Due to the smaller liposomes produced, we expect them to be less recognized by macrophages and remain in the circulatory system longer. Additionally, only safe solvents, water, and ethanol have been used in this study. In contrast, harmful organic solvents have been employed in other studies. Table 2 Comparing the results of this work with other article which produced irinotecan liposome Articles Mean size (nm) EE (%) Solvent and Chemical component [ 41 ] 116 98 Ethanol/ Triethylamine/ Sodium sucrose octasulfate [ 42 ] 198 91 Ammonium sulfate/ Citric acid/ Dehydrated alcohol/ NaOH [ 43 ] 69 94 Octadecylamine/ Chloroform/ Ammonium Sulfate [ 44 ] 150 ~ 90 Ethanol/ Triethanolamine/ CuSO 4 / H 2 SO 4 / Citrate buffer/ EDTA/ Sucrose/ HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) This work 55 95 Ethanol/Water 3.2. Storage stability study The storage stability of the IRH PEGylated liposomes made under ideal conditions was examined over 8 weeks. To assess the impact of time on the stability of the manufactured liposomes, they were stored at 4°C in the dark. At various time intervals, the mean diameter of liposomes and their EE were examined (Fig. 5d). As seen in Fig. S4d, the initial EE and particle size for PEGylated liposomes were found to be 93.8% and 55 nm, respectively. After 2 months, the EE reduced to 91.3% while the mean size of IRH PEGylated liposomes reached 61 nm. The experiment showed minimal change in initial IRH PEGylated liposomes kept at 4°C and approved these liposomes were stable for up to 60 days with no discernible changes in size and EE. This occurrence happened because liposomes were thermodynamically stable due to the homogeneous charge distribution on their surface. However, a slight change in liposome size resulted from their breakdown. Furthermore, even after storage, the spherical liposomes were still visible in the FESEM image (Fig. 5c). These results also demonstrated the high stability of the produced IRH liposomes. 3.3. Irinotecan hydrochloride release test To determine how liposomes affected the kinetics of drug release, drug release experiments were carried out at 37 C. Figure 6 displays the release profile of IRH from PEGylated and conventional liposomes and the Free Drug as a Control. According to the results, drug penetration through the dialysis membrane was not a limiting step because the release of free IRH was rapid, and 97% of the drug was released over 5 hours. On the other hand, during the initial sampling period (120 min), it was discovered that both PEGylated and conventional liposomes had an initial burst in the drug release that was greater than 18% and 10% for non-pegylated and pegylated liposomes, respectively. The observed burst release may be due to the release of the IRH that was adsorbed on the surface of the liposome. A steady drug release was observed following the first burst release, and over 10 hours, about 22 and 46% of the drug from PEGylated and traditional liposomes were released. The dissolved medication within the liposome's core diffusing into the dissolution medium may cause this delayed release. Moreover, the release rate of irinotecan from PEGylated liposomes was much lower than that from non-PEGylated Liposomes because of the more rigid bilayers of PEGylated Liposomes. 4. Conclusion This work used the ESSAS method to generate PEGylated liposomes of the powerful anticancer medication irinotecan hydrochloride. The Box-Behnken design was used to analyze how temperature, pressure drop, and flow rate affected the properties of the IRH liposomes and identify the optimum condition. It was discovered that the model developed in this study could accurately predict the experimental data. Furthermore, it was found that the pressure drop of 29.9 MPa, temperature of 42°C, and flow rate of 2.4 mL/min were the ideal conditions for the production of IRH PEGylated liposomes. Under ideal circumstances, liposomes with EE = 94.6%, count = 758, and dimension = 55 nm were created. Besides that, the flow rate had a significant impact on the EE, and the pressure drop was the factor that had the biggest impact on the count and size of the PEGylated liposomes. These findings showed that, in contrast to most traditional methods, this technique produced smaller IRH liposomes with high EE and without harmful solvents. Furthermore, the ESSAS process requires one step and does not require further extrusion or ultrasonication phases. As a result, this technology is a quick and easy substitute for conventional methods to produce liposomes with the best and most controllable physicochemical features (size) while also being able to be applied in large-scale industrial applications and adaptable to an industrial GMP process. In the future, liposomes containing both hydrophobic and hydrophilic active compounds will be produced using this technique. Furthermore, modified coatings of liposomes will be investigated to create targeted-release vesicles. Declarations Author contributions M.M.: Methodology, doing experiment, writing original draft. M.K.: design, editing text, interpret the data. F.R.: Supervise the project. Funding This study was funded by Shahid Beheshti University (Grant No.1398). Competing interests: The authors declare no competing interests. Data Availability All data generated or analysed during this study are included in this published article Ethics approval Not applicable. Consent to participate Not applicable. Consent to publish All authors agreed with the content and give their consent to submit the manuscript for publication. Authors and Affiliations All authors: Department of Analytical Chemistry and pollutants, Shahid Beheshti University, Tehran, Iran References Yang T, Choi M-K, Cui F-D, Kim J S, Chung S-J, Shim C-K,Kim D-D. Preparation and evaluation of paclitaxel-loaded PEGylated immunoliposome. JCR. 2007; https://doi.org/10.1016/j.jconrel.2007.05.011 . Choi S, Kang B, Yang E, Kim K, Kwak M K, Chang P-S,Jung H-S. Precise control of liposome size using characteristic time depends on solvent type and membrane properties. Scientific Reports 2023; https://doi.org/10.1038/s41598-023-31895-z . Trucillo P, Campardelli R, Scognamiglio M,Reverchon E. Control of liposomes diameter at micrometric and nanometric level using a supercritical assisted technique. J. CO2 Util. 2019; https://doi.org/10.1016/j.jcou.2019.04.014 . Liu D, Cohen J,Turkman N. PEG2000-DBCO surface coating increases intracellular uptake of liposomes by breast cancer xenografts. Sci. Rep. 2022; https://doi.org/10.1038/s41598-022-14947-8 . Atyabi F, Farkhondehfai A,Esmaeili F. Preparation of pegylated nano-liposomal formulation containing SN-38: in vitro characterization and in vivo biodistribution in mice. Acta pharmaceutica 2009; Https://doi.org/10.2478/v10007-009-0020-0 . Dadashzadeh S, Mirahmadi N, Babaei M H,Vali A M. Peritoneal retention of liposomes: Effects of lipid composition, PEG coating and liposome charge. JCR. 2010; https://doi.org/10.1016/j.jconrel.2010.08.026 . Nakamura K, Yamashita K, Itoh Y, Yoshino K, Nozawa S,Kasukawa H. Comparative studies of polyethylene glycol-modified liposomes prepared using different PEG-modification methods. Biochim. Biophys. Acta - Biomembr. 2012; https://doi.org/10.1016/j.bbamem.2012.06.019 . Syama K, Jakubek Z J, Chen S, Zaifman J, Tam Y Y C,Zou S. Development of lipid nanoparticles and liposomes reference materials (II): cytotoxic profiles. Sci. Rep. 2022; https://doi.org/10.1038/s41598-022-23013-2 . Mirhadi E et al. Redox-sensitive doxorubicin liposome: a formulation approach for targeted tumor therapy. Sci. Rep. 2022; https://doi.org/10.1038/s41598-022-15239-x . Vali A M, Toliyat T, Shafaghi B,Dadashzadeh S. Preparation, Optimization, and Characterization of Topotecan Loaded PEGylated Liposomes Using Factorial Design. Drug Dev. Ind. Pharm. 2008; https://doi.org/10.1080/03639040701385055 . Hashemzadeh H, Javadi H,Darvishi M H. Study of Structural stability and formation mechanisms in DSPC and DPSM liposomes: A coarse-grained molecular dynamics simulation. Sci. Rep. 2020; https://doi.org/10.1038/s41598-020-58730-z . Carrion C, Domingo J C,de Madariaga M A. Preparation of long-circulating immunoliposomes using PEG–cholesterol conjugates: effect of the spacer arm between PEG and cholesterol on liposomal characteristics. Chem. Phys. Lipids. 2001; https://doi.org/10.1016/S0009-3084(01)00178-5 . Zhang J et al. The preparation, characterization of Lupeol PEGylated liposome and its functional evaluation in vitro as well as pharmacokinetics in rats. Drug Dev. Ind. Pharm. 2019; https://doi.org/10.1080/03639045.2019.1569038 . Wei H et al. Active loading liposomal irinotecan hydrochloride: Preparation, in vitro and in vivo evaluation. Asian Journal of Pharmaceutical Sciences 2013; https://doi.org/10.1016/j.ajps.2013.10.006 . Xiao H,Sedlařík V. A Rapid and Sensitive HPLC Method for Simultaneous Determination of Irinotecan Hydrochloride and Curcumin in Co-delivered Polymeric Nanoparticles. Journal of Chromatographic Science 2020; https://doi.org/10.1093/chromsci/bmaa033 . Rajpoot K,Jain S K. Irinotecan hydrochloride trihydrate loaded folic acid-tailored solid lipid nanoparticles for targeting colorectal cancer: development, characterization, and in vitro cytotoxicity study using HT-29 cells. Journal of Microencapsulation 2019; https://doi.org/10.1080/02652048.2019.1665723 . Poudel B K et al. Development of polymeric irinotecan nanoparticles using a novel lactone preservation strategy. Int. J. Pharm. 2016; https://doi.org/10.1016/j.ijpharm.2016.08.018 . Chen Y et al. Pharmacovigilance of herb-drug interactions: A pharmacokinetic study on the combination administration of herbal Kang’ai injection and chemotherapy irinotecan hydrochloride injection by LC–MS/MS. Journal of Pharmaceutical and Biomedical Analysis 2021; https://doi.org/10.1016/j.jpba.2020.113784 . Naik S, Patel D, Surti N,Misra A. Preparation of PEGylated liposomes of docetaxel using supercritical fluid technology. J. Supercrit. Fluids. 2010; https://doi.org/10.1016/j.supflu.2010.02.005 . Trucillo P,Reverchon E. Production of PEG-coated liposomes using a continuous supercritical assisted process. The Journal of Supercritical Fluids 2021; https://doi.org/10.1016/j.supflu.2020.105048 . Penoy N, Grignard B, Evrard B,Piel G. A supercritical fluid technology for liposome production and comparison with the film hydration method. Int. J. Pharm. 2021; https://doi.org/10.1016/j.ijpharm.2020.120093 . Harbi I, Aljaeid B, El-Say K M,Zidan A S. Glycosylated Sertraline-Loaded Liposomes for Brain Targeting: QbD Study of Formulation Variabilities and Brain Transport. AAPS PharmSciTech 2016; https://doi.org/10.1208/s12249-016-0481-7 . Ahangari H, King J W, Ehsani A,Yousefi M. Supercritical fluid extraction of seed oils – A short review of current trends. Trends Food Sci. Technol. 2021; https://doi.org/10.1016/j.tifs.2021.02.066 . Chauhan T et al. Negatively charged liposomes of sertraline hydrochloride: Formulation, characterization and pharmacokinetic studies. Journal of Drug Delivery Science and Technology 2020; https://doi.org/10.1016/j.jddst.2020.101780 . Maja L, Željko K,Mateja P. Sustainable technologies for liposome preparation. The Journal of Supercritical Fluids 2020; https://doi.org/10.1016/j.supflu.2020.104984 . Salehi H, Karimi M,Raofie F. Micronization and coating of bioflavonoids extracted from Citrus sinensis L. peels to preparation of sustained release pellets using supercritical technique. JICS. 2021; https://doi.org/10.1007/s13738-021-02262-4 . Santo I E, Campardelli R, Albuquerque E C, de Melo S V, Della Porta G,Reverchon E. Liposomes preparation using a supercritical fluid assisted continuous process. Chemical Engineering Journal 2014; https://doi.org/10.1016/j.cej.2014.03.099 . Karimi M, Raofie F,Karimi M. Production Ganoderma lucidum extract nanoparticles by expansion of supercritical fluid solution and evaluation of the antioxidant ability. Scientific Reports 2022; https://doi.org/10.1038/s41598-022-13727-8 . Sharifi F, Zhou R, Lim C, Jash A, Abbaspourrad A,Rizvi S S H. Generation of liposomes using a supercritical carbon dioxide eductor vacuum system: Optimization of process variables. Journal of CO2 Utilization 2019; https://doi.org/10.1016/j.jcou.2018.12.011 . Mohammadi M, Karimi M,Raofie F. Expansion supercritical fluid into an aqueous solution (ESSAS), a new technique for creating nano-size cyanocobalamin-loaded liposomes, and optimization of involved parameters. JICS. 2023; https://doi.org/10.1007/s13738-023-02930-7 . Yousefi M, Rahimi-Nasrabadi M, Pourmortazavi S M, Wysokowski M, Jesionowski T, Ehrlich H,Mirsadeghi S. Supercritical fluid extraction of essential oils. TrAC Trends in Analytical Chemistry 2019; https://doi.org/10.1016/j.trac.2019.05.038 . Tavares Luiz M et al. Design of experiments (DoE) to develop and to optimize nanoparticles as drug delivery systems. European Journal of Pharmaceutics and Biopharmaceutics 2021; https://doi.org/10.1016/j.ejpb.2021.05.011 . Jacyna J, Kordalewska M,Markuszewski M J. Design of Experiments in metabolomics-related studies: An overview. Journal of Pharmaceutical and Biomedical Analysis 2019; https://doi.org/10.1016/j.jpba.2018.11.027 . Aslan N,Cebeci Y. Application of Box–Behnken design and response surface methodology for modeling of some Turkish coals. Fuel. 2007; https://doi.org/10.1016/j.fuel.2006.06.010 . Bezerra M A, Santelli R E, Oliveira E P, Villar L S,Escaleira L A. Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta 2008; https://doi.org/10.1016/j.talanta.2008.05.019 . Salehi H, Karimi M, Rezaie N,Raofie F. Extraction of β-Carboline alkaloids and preparation of extract nanoparticles from Peganum harmala L. capsules using supercritical fluid technique. Journal of Drug Delivery Science and Technology 2020; https://doi.org/10.1016/j.jddst.2020.101515 . Varona S, Martín Á,Cocero M J. Liposomal Incorporation of Lavandin Essential Oil by a Thin-Film Hydration Method and by Particles from Gas-Saturated Solutions. Industrial & Engineering Chemistry Research 2011; https://doi.org/10.1021/ie102016r . Lévai G, Martín Á, de Paz E, Rodríguez-Rojo S,Cocero M J. Production of stabilized quercetin aqueous suspensions by supercritical fluid extraction of emulsions. J. Supercrit. Fluids. 2015; https://doi.org/10.1016/j.supflu.2015.02.019 . Liu S, Yang F, Zhang C, Ji H, Hong P,Deng C. Optimization of process parameters for supercritical carbon dioxide extraction of Passiflora seed oil by response surface methodology. J. Supercrit. Fluids. 2009; https://doi.org/10.1016/j.supflu.2008.09.013 . Jiao Z, Wang X, Han S, Zha X,Xia J. Preparation of vitamin C liposomes by rapid expansion of supercritical solution process: Experiments and optimization. Journal of Drug Delivery Science and Technology 2019; https://doi.org/10.1016/j.jddst.2019.02.015 . Wang T, He W, Du Y, Wang J,Li X. Redox-sensitive irinotecan liposomes with active ultra-high loading and enhanced intracellular drug release. Colloids and Surfaces B: Biointerfaces 2021; https://doi.org/10.1016/j.colsurfb.2021.111967 . Zhang Z,Yao J. Preparation of Irinotecan-Loaded Folate-Targeted Liposome for Tumor Targeting Delivery and Its Antitumor Activity. AAPS PharmSciTech 2012; https://doi.org/10.1208/s12249-012-9776-5 . Zhang L, Cao D Y, Wang J, Xiang B, Dun J N, Fang Y,Xue G Q. PEG-coated irinotecan cationic liposomes improve the therapeutic efficacy of breast cancer in animals. Eur Rev Med Pharmacol Sci 2013; Hattori Y, Shi L, Ding W, Koga K, Kawano K, Hakoshima M,Maitani Y. Novel irinotecan-loaded liposome using phytic acid with high therapeutic efficacy for colon tumors. J Control Release 2009; https://doi.org/10.1016/j.jconrel.2009.01.013 . Additional Declarations No competing interests reported. Supplementary Files Supplementary.docx Cite Share Download PDF Status: Published Journal Publication published 05 Sep, 2024 Read the published version in Discover Nano → Version 1 posted Editorial decision: Revision requested 13 Jun, 2024 Reviews received at journal 09 Jun, 2024 Reviewers agreed at journal 04 Jun, 2024 Reviews received at journal 04 Jun, 2024 Reviewers agreed at journal 04 Jun, 2024 Reviewers invited by journal 04 Jun, 2024 Editor assigned by journal 03 Jun, 2024 Submission checks completed at journal 03 Jun, 2024 First submitted to journal 28 May, 2024 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-4492900","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":314011114,"identity":"ae61b119-b3d8-4143-abcd-207e7bef150e","order_by":0,"name":"Misagh Mohammadi","email":"","orcid":"","institution":"Shahid Beheshti University","correspondingAuthor":false,"prefix":"","firstName":"Misagh","middleName":"","lastName":"Mohammadi","suffix":""},{"id":314011115,"identity":"68278de7-8fdb-4e1b-b9b8-ad7980ea0f44","order_by":1,"name":"Mehrnaz Karimi","email":"","orcid":"","institution":"Shahid Beheshti University","correspondingAuthor":false,"prefix":"","firstName":"Mehrnaz","middleName":"","lastName":"Karimi","suffix":""},{"id":314011116,"identity":"262d12aa-f68b-4db5-bac7-1195130f8591","order_by":2,"name":"Farhad Raofie","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYBAC9gYQaSPBwM/AAxFhI6SF5wCITJNgkGwgUQsDg8EBHiIdxsPA+/DDhwQLOePzZ49JMNTYMfBJHyCkhd1YckaChLHZjTyg844lM7DxJeDXYg90uzTvD4nEbTd4zCQY2A4wsBFyIA8DG/NvngSJ+s39Z4Ba/hGnhU0aqCXBgCHHTIKxjUgtlkC/GM64kZdskdiXzEOUw258SKiT5+8/e/DGh292cvI9BLQwyD9A4iQwMBAbO6NgFIyCUTAK8AEAd5gwIWw+B1sAAAAASUVORK5CYII=","orcid":"","institution":"Shahid Beheshti University","correspondingAuthor":true,"prefix":"","firstName":"Farhad","middleName":"","lastName":"Raofie","suffix":""}],"badges":[],"createdAt":"2024-05-28 19:40:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4492900/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4492900/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s11671-024-04071-z","type":"published","date":"2024-09-05T16:08:24+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58346045,"identity":"d5a22504-73cb-48e7-bf8b-5f92345a59fa","added_by":"auto","created_at":"2024-06-14 07:48:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":91178,"visible":true,"origin":"","legend":"\u003cp\u003ethe schematic diagram of the ESS set up: (1) CO2 Cylinder, (2) Chiller, (3) High-pressure pump unit, (4) Heat exchanger, (5) Oven, (6) Equilibration vessel, (7) Restrictor, (8) Production vessel.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4492900/v1/fe0d8ba0a562f04f88a29bb2.png"},{"id":58345474,"identity":"546a97d6-8cf4-4d54-917b-a7afc62d0208","added_by":"auto","created_at":"2024-06-14 07:40:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":80809,"visible":true,"origin":"","legend":"\u003cp\u003eStandardized Pareto chart for (a) Size, (b) Count, (c) EE of produced irinotecan PEGylated liposomes, and main effect diagram for (d) Size, (e) Count, and (f) EE of produced irinotecan PEGylated liposomes, respectively.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4492900/v1/d3842bde6cb5f6087efdc493.png"},{"id":58346046,"identity":"448a55bc-a697-4598-8878-fca4e1642d73","added_by":"auto","created_at":"2024-06-14 07:48:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1703810,"visible":true,"origin":"","legend":"\u003cp\u003eFESEM images of produced irinotecan PEGylated liposomes (a, c, e, g, i, and k) experiments 1, 2, 5, 11, 13, and 14 respectively. Corresponding liposome size distribution (b, d, f, h, j, and l) experiments 1, 2, 5, 11, 13, and 14 respectively.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4492900/v1/8563659c638046d821252507.png"},{"id":58345480,"identity":"c51392fa-bee7-470f-aa94-260e7214824c","added_by":"auto","created_at":"2024-06-14 07:40:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":327684,"visible":true,"origin":"","legend":"\u003cp\u003eEstimated response surface for (a,d,g) pressure drop was fixed at 22.5 MPa, (b,f,h) flow rate was fixed at 2 mL/min, (c,e,i) temperature was fixed at 52.5 ˚C for the mean size, Count, and EE, respectively.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4492900/v1/62dd578ad4cb1e7da9a8549e.png"},{"id":58346908,"identity":"bcd372b0-7626-48b8-95c8-5a3080e55e0e","added_by":"auto","created_at":"2024-06-14 07:56:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":595069,"visible":true,"origin":"","legend":"\u003cp\u003ea) FESEM images of produced irinotecan liposome at optimal condition, b) produced liposome size distribution at optimal condition, c) FESEM images of produced irinotecan liposome after 8 week, and d) mean Diameter, and EE stability test of Pegylated liposomes loaded with irinotecan hydrochloride\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4492900/v1/1d0363c6b01a15d2c3b4f4e1.png"},{"id":58345476,"identity":"25373344-e756-4d55-a977-79b17bcd2fce","added_by":"auto","created_at":"2024-06-14 07:40:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":82259,"visible":true,"origin":"","legend":"\u003cp\u003eDrug release test of irinotecan hydrochloride from PEGylated liposomal formulation, traditional liposomes, and pure irinotecan hydrochloride solution as control at different periods of time.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4492900/v1/ec2fa3e817a5ec603f09e632.png"},{"id":64186203,"identity":"c498322e-e2d7-47db-b702-e31d9c6e778d","added_by":"auto","created_at":"2024-09-09 16:25:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3584254,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4492900/v1/5837524e-1eb4-455d-a51a-c7259d376a47.pdf"},{"id":58345479,"identity":"6a3270a7-092f-4a1a-b1dd-4ab3ef46f2ab","added_by":"auto","created_at":"2024-06-14 07:40:32","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1660409,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-4492900/v1/01ad136387f0d0cf1e0d6b64.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Preparation Irinotecan Hydrochloride Loaded PEGylated Liposomes Using Novel Method Supercritical Fluid and Condition Optimized by Box-Behnken Design","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNanotechnology has recently begun to develop in various fields, including cosmetics, pharmaceuticals, medicine, food, and environmental research. Most pharmaceutical research has focused on drug delivery systems (DDS). Developing nanocarriers, such as lipid carriers (known as liposomal formulations), is one of the most promising strategies for drug delivery systems, as they significantly improve drug pharmacokinetics and, in turn, reduce toxicity and improve therapeutic efficacy and biocompatibility of drugs [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These spherical vesicles are composed of one or more phospholipid bilayers. These carriers are biocompatible because they resemble human cells whose exterior lipid bilayer encloses the aqueous compartment [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMoreover, liposomes are advantageous drug carriers because they can constantly release the captured substrates, which include both hydrophilic and hydrophobic substrates [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Liposomes can also provide a slow drug release, resulting in long-term drug exposure to tumor cells and improving their efficacy[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The mononuclear phagocyte system (MPS), which ingests and eliminates liposomes circulating in the bloodstream, is assumed to be the mechanism by which liposomes are cleared from the body. So, it is especially crucial to prevent capture by the liver's and spleen's phagocytic cells to prolong blood circulation time. Chain length, lipid unsaturation, lipid composition, size, and Zeta potential are just a few of the liposome properties that impact how long they circulate through the blood [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent research has shown that some liposome formulations, such as those with a small amount of phospholipid modified by polyethylene glycol (PEG), allow these carriers to remain in the blood for longer periods [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Although the specific mechanism of the MPS avoidance phenomenon is unknown, it is thought that the PEG chains, due to their water-binding ability and flexibility, prevent the uptake of opsonizing proteins that guide liposomes to macrophages [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Furthermore, PEG can protect liposomes by producing a polymer layer on the liposome surface, increasing the hydrophilicity of the surface, increasing the mutual repulsion between the polymer coating and blood components, and shielding surface charge [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Also, through an increased permeability and retention (EPR) effect via a passive targeting mechanism, liposomes modified with polyethylene glycol spontaneously accumulate in solid tumors [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. PEGylated liposomes offer a desirable platform for enhancing the therapeutic index of a range of anticancer medications [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIrinotecan hydrochloride (IRH), a semisynthetic derivative of camptothecin, is frequently used in treating colorectal cancer (CRC) such as colorectal adenocarcinoma and malignancies related to small cells in the lung [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. IRH has been found to specifically engage with the topoisomerase, which controls DNA topology and carries out several nuclear functions, including DNA replication, recombination, and repair. So, it can prevent DNA replication in tumor cells in the case of CRC [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Currently, the use of this substance as a single agent or in combination with other chemotherapeutic drugs is approved for treating colorectal, ovarian, cervical, and small-cell lung cancer [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. But it has been found that IRH has dosage-dependent side effects such as myelosuppression and severe diarrhea because of the bis-piperidine group, which is known to constitute dose-limiting toxicity and restrict the dose that can be used safely [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, due to the extremely short half-life of IRH, continuous infusions are necessary to maintain its therapeutically effective blood level, which can lead to cumulative toxicities. Because of these drawbacks, effective delivery strategies can both (a) avoid the hydrolytic conversion of the lactone configuration of the E-ring to an inactive open-ring carboxylate and (b) provide prolonged IRH release have been developed [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. One of the most efficient drug delivery strategies is the liposomal formulation. Current studies claim that IRH is favorable to effective encapsulation in pharmaceutically viable liposome systems due to its physicochemical properties. These liposomal formulations can decrease IRH in vivo clearance and boost anticancer effectiveness [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In 2015, the irinotecan liposome injectable Onivyde received US FDA (Food and Drug Administration) approval to treat patients with advanced pancreatic cancer [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLiposomes can be made using several methods, with at least 14 major methods reported. Lipid film hydration, also known as thin-layer evaporation, rehydration-dehydration, reverse-phase evaporation, ethanol injection, French press, ether infusion, and detergent dialysis techniques, are the seven most widely used methods [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These techniques have various drawbacks, such as difficult control of particle size distribution, low encapsulation efficiency, high liposome size, and low method reproducibility due to discontinuous layouts [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Moreover, the methods above require a large value of organic solvents, which are harmful to the environment and the human body. Due to the need to completely remove any leftover organic solvent, the use of organic solvents in manufacturing drug delivery carriers must be avoided or minimized as much as feasible [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Although there is a way to create liposomes by repeatedly freezing and melting an aqueous solution, it is evident that this method requires a lot of energy. So, these techniques are unsuitable for producing liposomes in large quantities because they involve numerous steps and cannot fully overcome those limitations [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSupercritical fluid (SCF) has been considered in producing nanoliposomes to reduce or eliminate the use of organic solvents and achieve greener chemistry [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. When a fluid is in the supercritical state, it combines the physicochemical characteristics of both liquids and gases, including high density and viscosity. These characteristics are adjustable with changes in pressure and temperature [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The most widely used SCF is carbon dioxide because it is cheap, nontoxic, and has a low critical temperature (31.1\u0026deg;C) and pressure (7.38 MPa), making it safe and suitable for handling pharmaceutical chemicals. Moreover, after depressurization, CO\u003csub\u003e2\u003c/sub\u003e reverts to its gaseous state, It prevents any problem caused by the toxicity of residual solvents in the final product [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In contrast to conventional ones, these processes are quick,easy, and create liposomes with nanometric dimensions; as a result, the need for post-processing steps like extrusion or sonication is frequently dropped.\u003c/p\u003e \u003cp\u003eMoreover, liposomes are produced with greater physicochemical property control and reproducibility, making these methods suitable for industrial production under GMP (Good manufacturing practice) guidelines. In addition, due to the properties of SCF, the use of organic solvents can be controlled or eliminated [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. SCF can be used as an antisolvent in the SAS (Supercritical antisolvent), ASES (Aerosol solvent extraction system), or GAS (Gas Antisolvent) processes, in which an organic solvent is utilized to solubilize lipids, which are subsequently precipitated by the antisolvent effect of SCF. Moreover, The SCF could be used as a dispersing agent. These techniques involve dispersing the lipids in pure water, followed by applying SCF pressure to cause fine dispersion of the lipids due to collision and shear forces [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In this procedure, the phospholipids are evenly diffused into the medium, and liposomes are generated during depressurization. Moreover, another option is that SCF operates as a lipid solvent, such as Rapid Expansion of Supercritical Solution (RESS), supercritical Assisted liposome formation (SuperLip), and Expansion Supercritical Fluid into an aqueous solution (ESSAS) [\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn general, the effectiveness of the supercritical fluid's ability to generate nano-liposomes depends on several factors, including flow rate, pressure, time, temperature, etc. In the circumstances like this, when many factors may affect the final result, optimizing and modeling process variables using approaches such as the one factor at a time (OFAT) or design of experiments (DOE) approach is an excellent way to increase process efficiency [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Since in the DOE strategy, all the interactions between various parameters are considered, it produces more accurate findings than the OFAT strategy. In addition, DOE requires fewer experiments to achieve ideal conditions, so this method is more valuable than the single-factor method [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. One of the DOEs is the Behnken box (BB) design, created by combining an incompletely balanced or semi-balanced block design with a two-level factorial design in a special way and enables the rapid estimation of first- and second-order coefficients of the mathematical mode [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Furthermore, the three-component Box-Behnken matrix has a spherical, rotatable design, which means that all design points (aside from the center) are dispersed uniformly across the sphere [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis research aims to develop irinotecan hydrochloride PEG-coated liposomes using the ESSAS method previously introduced by our team [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. ESSAS is a modified version of RESS and supercritical fluid extraction of emulsions [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] in which the pressure suddenly drops, but it remains higher than the critical pressure. As a result, phospholipids' precipitation rate can be controlled, and homogeneous liposomes with narrow size distribution can be produced. Furthermore, whereas in other processes, the pressure is brought down to atmospheric pressure from above the critical pressure, this procedure allows for producing massive liposomes with a depressurization rate of fewer than 10 MPa. Also, we employ BB to research and optimize the variables influencing nanoliposome size, encapsulation efficiency, the number of nanoliposomes formed, and vesicle stability. Measurements will be made using the Zeta potential and the Field Emission Electron Microscope (FESEM) to show the stability, size, and morphology of the generated liposomes. To confirm the PEG-coating's success and manage drug release kinetics, the release of irinotecan hydrochloride will be investigated.\u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1.Materials\u003c/h2\u003e \u003cp\u003eCholesterol, m-dPEG\u0026reg;\u003csub\u003e12\u003c/sub\u003e-amido-dPEG\u0026reg;\u003csub\u003e24\u003c/sub\u003e-DSPE (mPEG\u003csub\u003e1500\u003c/sub\u003e-DSPE), and 1,2-Distearoyl-sn-glycerol-3- phosphocholine (DSPC) were purchased from Sigma-Aldrich. Carbon dioxide with a purity of more than 99.9% was supplied by Roham Co. in Tehran, Iran. Moreover, Aventis Pharma (\u003cem\u003eMumbai\u003c/em\u003e, India) provided the Irinotecan hydrochloride standard used to encapsulate. HPLC-grade ethanol and methanol were obtained from Caledon (Georgetown, Ont., Canada). Furthermore, all formulations were created using distilled and deionized water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2.Preparation of Nanoliposomes\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. Preparation of the phospholipid cargo solution\u003c/h2\u003e \u003cp\u003eAt 25\u0026deg;C, 120 mg DSPC and 30 mg mPEG1500-DSPE were dissolved in a 40 mL mixture of ethanol/water (30% V/V), and the desired solution was then supplemented with 25 mg cholesterol and 30 mg irinotecan hydrochloride standard. After that, the solution of the active ingredients was then stirred for 90 minutes at 1100 rpm and afterward, 2 mL of the resulting solution was transferred to the equilibrium vessel for liposome preparation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2. Nanoliposome production process\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this investigation, the expansion Supercritical Fluid into an aqueous solution (ESSAS) approach, was utilized to create liposomes on a Suprex automated Prepmaster 44 system (AP44) with a variable flow restrictor (Pittsburgh, PA). Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e depicts a schematic of the ESSAS apparatus used to create liposomes. Three valves and two high-pressure vessels, referred to as the equilibration and production vessels, with inner volumes of 10 cm\u003csup\u003e3\u003c/sup\u003e and dimensions of 16 mm i.d and 141 mm height, formed the device's overall layout. Also, the vessels' temperatures were tracked using a thermocouple housed inside the oven. A digital pressure gauge was used to assess the pressure inside the high-pressure vessels.\u003c/p\u003e \u003cp\u003eTo create PEGylated liposomes, 2 mL of the cargo solution was first moved to the equilibration vessel, and 7 mL of HPLC-grade water was added to the production vessel. To achieve the highest EE, the greatest number of produced liposomes, and the smallest size of vesicles, the parameters of temperature, flow rate, and pressure drop (Pd), whose range were determined by the initial tests, were studied using Statgraphics XVII software and Box-Behnken designs. Furthermore, the collection time, equilibrium pressure, and equilibrium time were held constant for all experiment runs at 15 minutes, 40 MPa, and 30 minutes, respectively. Because the solubility of supercritical fluid increases with increasing pressure, and the equilibrium pressure was set at the highest operating pressure of the device at 40 MPa. Also, after 15 minutes, all the solution in vessel1 was transferred to vessel 2, and higher collection times were practically useless. In the first stage, by opening valve 1, the condensed carbon dioxide produced by the high-pressure pump was transferred to the equilibrium vessel through a capillary tube. The cargo solution was allowed to dissolve in carbon dioxide for 30 minutes once the pressure hit 40 MPa and the temperature reached the value determined by the software. After the equilibrium time was up, valves 2 and 3 were opened to create the pressure drop, and the contents of the equilibrium vessel were expanded into the production vessel while supercritical CO\u003csub\u003e2\u003c/sub\u003e (SC-CO\u003csub\u003e2\u003c/sub\u003e) was passed from it. At this stage, a sudden pressure drop occurred. As a result, the density and solubility of SC-CO\u003csub\u003e2\u003c/sub\u003e decreased, and using nucleation, substances dissolved in SC-CO\u003csub\u003e2\u003c/sub\u003e began to precipitate in the production vessel. Eventually, during this procedure, the precipitated phospholipids, mPEG\u003csub\u003e1500\u003c/sub\u003e-DSPE, and cholesterol were brought together and spontaneously produced nanoliposomes containing a portion of IRH. The production vessel achieved ambient pressure when the operation was finished because valve 3 was left open while valves 1 and 2 were closed. The resultant solution was then collected in a vial and kept at 4 \u0026deg; C for future analysis.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3.Characterization of Liposomes: morphology and size distribution\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. Field Emission Scanning Electron Microscopy (FESEM)\u003c/h2\u003e \u003cp\u003eA field emission scanning electron microscope was used to investigate the size and morphology of liposomes (FESEM, MIRA3 TESCAN-XMU, and the Czech Republic). In specifics, two droplets of the nanoliposome suspension were sprayed on the aluminum bases and allowed to air dry. Then, a layer of platinum was applied to dry liposome suspension using a sputtering apparatus (Pelco SC-7, Ted Pella Inc., Redding, CA). The liposomes were subsequently seen using an accelerating voltage of up to 10 kV at a magnification of 250 and 90,000 times. The images were analyzed using the ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2. Size distribution, and Zeta potential of liposomes\u003c/h2\u003e \u003cp\u003eFollowing the ESSAS process, Dynamic Light Scattering (mod. Zetasizer Nano S, United Kingdom) was used to measure the mean diameter (MD) and zeta potential (ZP) of the generated nanoliposomes on each sample. This device has a 633 nm wavelength, 5.0 mW He-Ne laser with a 173-degree scattering angle. Each test required 1 mL of the nanoliposome suspension, and measurements were made at room temperature in triplicates.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.4.Storage stability study\u003c/h2\u003e \u003cp\u003eThe storage stability of the nanoliposome suspension produced by the ESSAS method was examined for up to 8 weeks. At specific time intervals, the nanoliposome's EE and size were examined for storage stability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.5.Determination of Encapsulation Efficiency (EE)\u003c/h2\u003e \u003cp\u003eThe encapsulation efficiency of the manufactured PEGylated liposomes was investigated by measuring non-encapsulated IRH in the generated liposome suspension. We determined the amount of irinotecan hydrochloride entrapped in the nanoliposomes by comparing the initial amount of the drug used to make the cargo solution to the quantity of unencapsulated drug that remained in the liposome suspension after production. To do this, the liposome suspension created using the ESSAS approach was centrifuged at 4600 rpm for 75 minutes. After that, the supernatants were separated, and irinotecan hydrochloride concentrations were determined using high-performance liquid chromatography (HPLC).\u003c/p\u003e \u003cp\u003eA liquid chromatographer (HP Agilent 1050, Agilent, Santa Clara, California, USA) in conjunction with a variable wavelength detector and HP ChemStation software was used to carry out the HPLC method for determining and monitoring the irinotecan hydrochloride content in supernatants. The separation was performed using an Agilent HT Zorbax SB-C18 column (5 \u0026micro;m particle size, 4.6 mm 250 mm; Agilent Technologies, Santa Clara, CA) with 10 \u0026micro;L of sample injection volume. The mobile phase in the isocratic elution protocol was 3% V/V triethylammonium acetate (TEAA) buffer (pH 3) and acetonitrile (70:30 V/V). The mobile phase flow rate and detector wavelength were constant throughout the 25-minute analysis at 1 mL/min and 379 nm, respectively. Irinotecan hydrochloride standard curves were created using the drug standard at 10 to 500 ppm concentrations before analyzing the supernatants. The encapsulation efficiency is then assessed using Eq.\u0026nbsp;1, where CT denotes the initial amount of IRH and F denotes the amount of free irinotecan hydrochloride in the liposome suspension.\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\text{E}\\text{E}\\text{\\%} =\\frac{\\text{C}\\text{t} - \\text{F}}{\\text{C}\\text{t} } \\times 100\\text{\\%}\\)\u003c/span\u003e \u003c/span\u003e Eq.\u0026nbsp;(1)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.6.Drug release test\u003c/h2\u003e \u003cp\u003eDrug release tests were conducted at 37 \u0026deg; C using the HPLC procedure used to quantify the EE. For this purpose, A dialysis sack (MWCO 3500 Da, Sigma Aldrich, Milan, Italy) was filled with 4 mL of the generated IRH PEGylated liposomes and clamped on both sides. After that, it was placed in a 150 mL volume of distilled water and stirred at 300 rpm without any light. 1 mL aliquots of the dialysate (a distilled water bath solution containing the released IRH) were taken at fixed time intervals to calculate the quantity of drugs released and to indicate drug release profiles. Following the evaporation of the dialysate's solvent, the remaining irinotecan hydrochloride was dissolved in 200 \u0026micro;L of ethanol to raise the medication's concentration, and by the HPLC method amount of IRH was determined. The PEGylated liposomes that were produced under ideal circumstances were used for these studies. For comparison purposes, another release test was conducted using non-PEGylated vesicles (which were produced under the same conditions but without the presence of mPEG1500-DSPE) and pure irinotecan hydrochloride solution (10 mg drug in 10 mL water/ethanol 95% V/V).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003e3.1.Response Surface Methodology\u003c/h2\u003e\n\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\n\u003ch2\u003e3.1.1 Fitting the Model and Data Analysis\u003c/h2\u003e\n\u003cp\u003eThe main objective of producing PEGylated liposomes using the ESSAS method is to speed up the preparation and manufacturing processes while improving repeatability, encapsulation effectiveness, homogeneity, liposome count, and steady release of irinotecan for a more extended time compared to liposomes which is made by conventional methods. During the equilibrium time, the cargo solution compounds in vessel 1 were dissolved and saturated in the SC-CO\u003csub\u003e2\u003c/sub\u003e. The pressure was then quickly reduced during the production step by opening valves 2 and 3, allowing SC-CO\u003csub\u003e2\u003c/sub\u003e to flow into vessel 2. This caused the creation of fine water droplets and reduced the density and solvent power of SC-CO\u003csub\u003e2\u003c/sub\u003e. As a result, phospholipid, cholesterol, and mPEG\u003csub\u003e1500\u003c/sub\u003e-DSPE were precipitated from SC-CO\u003csub\u003e2\u003c/sub\u003e and quickly covered water droplets that had dissolved a portion of the irinotecan in themselves. This method led to the formation of reverse lipid micelles, which then fell into the aqueous bulk of vessel 2 and were stabilized by the second layer of phospholipids and mPEG\u003csub\u003e1500\u003c/sub\u003e-DSPE which were covering these structures, and finally, PEGylated Irinotecan liposomes were created. Nanoliposome characteristics, including EE, the number of liposomes produced at 1 \u0026micro;m scale, and liposome size, are influenced by temperature, flow rate, and pressure drop. Hence the parameters were optimized using the Box Behnken design, one of the experimental designs. According to the equation K\u0026thinsp;=\u0026thinsp;2K(K-1)\u0026thinsp;+\u0026thinsp;C, where k is the number of parameters, and C is the number of center point repetitions, 15 tests were finished with 3 center point repetitions. Also, the preliminary tests found that after 15 minutes, all of the cargo solution in vessel 1 is transferred into the production vessel. So, in all tests, the dynamic time was set at 15 minutes. Additionally, the equilibrium pressure was taken to be 40 MPa because the density of the supercritical fluid and, consequently, the solubility of the cargo solution compounds in it, increases with the increase of the equilibrium pressure. Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e displays the three factors with lower and higher design points in coded values. In addition, Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e displays the experimental conditions and the obtained outcomes. Analysis of responses seen throughout all runs was conducted using stat graphics software. It was discovered that a quadratic model provided the best fit. Additionally, the least squares method was used to compute the variance of this design and the model equation's coefficients. The results of ANOVA analysis of the experimental data for mean size, count, and EE are reported in Tables S2, S3, and S4, respectively. The significance of various coefficients, including linearity, quadraticity, and interaction, is listed in these tables along with the p-value, where the expressions of the model with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and p\u0026thinsp;\u0026gt;\u0026thinsp;0.05 denote significant and insignificant results, respectively.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eDesign matrix and the responses (BBD) for the production of irinotecan PEGylated nanoliposomes.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eExperiment\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFlow rate\u003c/p\u003e\n\u003cp\u003e(mL/min)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePressure drop\u003c/p\u003e\n\u003cp\u003e(MPa)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTemperature\u003c/p\u003e\n\u003cp\u003e(˚C)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMean size\u003c/p\u003e\n\u003cp\u003e(nm)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCount\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eEE\u003c/p\u003e\n\u003cp\u003e(%)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFigures\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRun 1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e52.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e53\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e368\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e86\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFigure\u0026nbsp;3a\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRun 2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e30.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e52.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e41\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e429\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e96\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFigure\u0026nbsp;3c\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRun 3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e65.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e57\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e170\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFigure S3a\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRun 4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e52.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e51\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e357\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e88\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFigure S3b\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRun 5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e65.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e74\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e64\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e81\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFigure\u0026nbsp;3g\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRun 6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e30.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e65.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e48\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e300\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e77\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFigure S3c\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRun 7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e52.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e49\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e350\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e87\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFigure S3d\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRun 8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e15.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e65.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e94\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e110\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFigure S3e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRun 9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e40.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e72\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e265\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e89\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFigure S3f\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRun 10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e30.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e40.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e710\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e85\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFigure S3g\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRun 11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e15.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e40.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e108\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e60\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e76\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFigure\u0026nbsp;3e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRun 12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e30.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e52.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e46\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e460\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e71\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFigure S3h\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRun 13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e40.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e180\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e76\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFigure\u0026nbsp;3i\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRun 14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e15.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e52.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e82\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e86\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e77\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFigure\u0026nbsp;3l\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRun 15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e15.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e52.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e98\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e60\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFigure S3i\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe formulas of the second-order polynomial equations resulting from the experimental designs were obtained as follows:\u003c/p\u003e\n\u003cp\u003eY\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;588.895\u0026ndash;42.125X\u003csub\u003e1\u003c/sub\u003e \u0026minus;\u0026thinsp;13.2333X\u003csub\u003e2\u003c/sub\u003e \u0026minus;\u0026thinsp;11.406X\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;3.0X\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;0.366667X\u003csub\u003e1\u003c/sub\u003eX\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;0.4X\u003csub\u003e1\u003c/sub\u003eX\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;0.226667X\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e \u0026minus;\u0026thinsp;0.0133333X\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;0.0992X\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e Eq.\u0026nbsp;(2)\u003c/p\u003e\n\u003cp\u003eY\u003csub\u003e2\u003c/sub\u003e = -3542.36\u0026thinsp;+\u0026thinsp;689.717 X\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;84.3167 X\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;80.714 X\u003csub\u003e3\u003c/sub\u003e \u0026minus;\u0026thinsp;112.417 X\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e \u0026minus;\u0026thinsp;1.9 X\u003csub\u003e2\u003c/sub\u003e \u0026minus;\u0026thinsp;3.82 X\u003csub\u003e1\u003c/sub\u003e X\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;0.228148 X\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e \u0026minus;\u0026thinsp;1.22667 X\u003csub\u003e2\u003c/sub\u003e X\u003csub\u003e3\u003c/sub\u003e \u0026minus;\u0026thinsp;0.487467 X\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e Eq.\u0026nbsp;(3)\u003c/p\u003e\n\u003cp\u003eY\u003csub\u003e3\u003c/sub\u003e = -45.58\u0026thinsp;+\u0026thinsp;2.95 X\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;2.78 X\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;3.564 X\u003csub\u003e3\u003c/sub\u003e \u0026minus;\u0026thinsp;3.625 X\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;0.766667 X\u003csub\u003e1\u003c/sub\u003eX\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;0.02 X\u003csub\u003e1\u003c/sub\u003eX\u003csub\u003e3\u003c/sub\u003e \u0026minus;\u0026thinsp;0.0644444 X\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e \u0026minus;\u0026thinsp;0.0186667 X\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e3\u003c/sub\u003e \u0026minus;\u0026thinsp;0.0328 X\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e Eq.\u0026nbsp;(4)\u003c/p\u003e\n\u003cp\u003eWhere Y\u003csub\u003e1\u003c/sub\u003e, Y\u003csub\u003e2\u003c/sub\u003e, and Y\u003csub\u003e3\u003c/sub\u003e stand for the mean size, count of PEGylated liposomes, and EE. Furthermore, the X\u003csub\u003e1\u003c/sub\u003e through X\u003csub\u003e3\u003c/sub\u003e stands for the flow rate (mL/min), pressure drop (MPa), and temperature (\u0026deg;C), respectively.\u003c/p\u003e\n\u003cp\u003eThe ANOVA results showed good R\u003csup\u003e2\u003c/sup\u003e and R\u003csup\u003e2\u003c/sup\u003e adjusted for mean size (97% and 91%), EE (98% and 96%), and the count of produced PEGylated irinotecan liposomes (99% and 97%), respectively. The high-adjusted R-square value demonstrates a strong correlation between the estimated and measured values, which verifies the accuracy of the data and the model. The \"Lack of Fit\" p-values for mean size, count, and EE were 0.0589, 0.0590, and 0.2544, respectively. As is clear, they are all more than 0.05, indicating that the Lack of Fit was not significant compared to the pure error and the model correctly predicted the experimental data. Additionally, Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e displayed how well the experimental size, count, and EE values fit the expected values. These outcomes demonstrate that the model created in this study can correctly project the experimental data.\u003c/p\u003e\n\u003cp\u003eIt should be noted that the zeta potential is used to measure the stability of liposomes in suspension; values higher than |30| mV indicate liposome stability. The PEGylated-liposomes generated exhibited good stability in this investigation, and the ZP was always greater than \u0026minus;\u0026thinsp;30 mV.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\n\u003ch2\u003e3.1.2. Pressure drop effect\u003c/h2\u003e\n\u003cp\u003eThe effect of each parameter on the characteristics of IRH PEGylated liposomes is shown using main effect graphs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee, and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ef) when one parameter is altered while the others are kept constant. additionally, the Pareto chart (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb, and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec) shows the parameters' impact and specifies which factors significantly impacted the responses at a 95% confidence level.\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed shows that increasing the pressure drop up to 28 MPa caused the IRH liposome size to fall from 86 to 41 nm before remaining constant. To explain this phenomenon, it can be said that a greater pressure drop caused a higher decrease in the density and solubility of SC-CO\u003csub\u003e2[19]\u003c/sub\u003e. As a result, it created a greater difference between the value of phospholipids that could be dissolved in SC-CO\u003csub\u003e2\u003c/sub\u003e before and after the expansion step, leading to higher supersaturation. According to the nucleation theory, more supersaturation encouraged nucleation during the expansion step, leading to smaller liposomes. Additionally, more nucleation caused the generation of more nuclei, so as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee, the count of produced liposomes increased.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs indicated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ef, the encapsulation efficiency of the created liposomes increased to 87.5% until the pressure drop reached at 27 MPa and decreased thereafter. Because as previously stated, increasing the pressure drop resulted in the production of more liposomes, which increased encapsulation efficiency. However, increasing the pressure drop resulted in the production of smaller liposomes with less internal space to trap IRH. As a result, the EE decreased due to the dominance of this effect on the number of liposomes after the pressure drop of 27 MPa. Figure\u0026nbsp;3(a, b, c, and d) show the FESEM picture and particle size distribution of experiments 1 and 2, indicating the effect of pressure drop on nanoliposome production. Furthermore, these figures demonstrate that spherical liposomes with a narrow size distribution were generated in this study. Moreover, the Pareto chart (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb, and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec) demonstrates that pressure drop significantly impacts the mean size, count, and EE of the production process.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\n\u003ch2\u003e3.1.3. Temperature effect\u003c/h2\u003e\n\u003cp\u003eThis study looked into the effects of temperatures from 40 to 65\u0026deg;C. According to Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb, and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec the mean size, count, and EE of liposomes were significantly influenced by temperature.\u003c/p\u003e\n\u003cp\u003eAccording to Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee, and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ef, the temperature had a reverse effect in this study. Firstly, the rising temperature caused more phospholipid soluble in CO\u003csub\u003e2\u003c/sub\u003e due to the increased mass transfer speed, leading to greater supersaturation [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. Therefore, the size of produced liposomes was reduced to 50 nm. Moreover, As the temperature rose, the energy of the molecules in the phospholipid bilayer increased. It improved the membrane's fluidity, which would encourage the generation of liposomes, make it easier for the drug to be wrapped around the wall material, and increase the amount of drug that can be loaded into each vesicle [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. So, the count and EE of liposomes rose to 370 and 87.5%, respectively.\u003c/p\u003e\n\u003cp\u003eOn the other hand, as the temperature increased, the density of SC-CO\u003csub\u003e2\u003c/sub\u003e decreased. As a result, it decreased the solubility of the compounds and the solvent capacity of SC-CO\u003csub\u003e2\u003c/sub\u003e, which led to lower supersaturation and the production of bigger (61 nm) and fewer liposomes (210) with lower EE (79%). The FESEM image and particle size distribution from experiments 5 and 11 are shown in Fig.\u0026nbsp;3e, 3f, 3g, and 3h, respectively. These results demonstrate how temperature affects the production of IRH PEGylated liposomes. These images' narrow and homogeneous particle size distribution suggested that uniform PEGylated liposomes were formed.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\n\u003ch2\u003e3.1.4. Flow rate effect\u003c/h2\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e shows that the flow rate in the 1 to 3 mL/min range did not significantly affect the mean size and number of liposomes. But the EE was highly influenced by this parameter. This was expected because pressure and temperature have a greater impact on SC-CO\u003csub\u003e2\u003c/sub\u003e properties such as density, mass transfer, and solubility than flow rate, which therefore have a greater effect on the size and number of liposomes produced. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee, and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ef, increasing the fluid flow rate resulted in higher mixing energy and greater atomization of cargo solution droplets in SC-CO\u003csub\u003e2\u003c/sub\u003e flow[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. These conditions caused liposomes to develop more quickly and around smaller droplets, leading to an increase in the number of liposomes and EE.\u003c/p\u003e\n\u003cp\u003eOn the other hand, after the flow rate reached 2.7 mL/min, the turbulence induced by the flow rate resulted in non-uniformity and the creation of larger liposomes, which also had larger internal space. As a result, the encapsulation efficiency remained unchanged. Figures\u0026nbsp;3i, 3j, 3k, and 3l, display the FESEM image and the particle size distribution of experiments 13 and 14, respectively. These figures show how flow rate influences the morphology, count, and homogeneity of IRH liposomes.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.5. Contour plots and Response Surface analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAs functions of pressure drop, temperature, and flow rate, the response surface diagrams and contour plots of size, count, and EE of PEGylated liposomes were achieved by adjusting the two of them while the other one held constant, which is shown in\u003c/em\u003e Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e and S2, \u003cem\u003erespectively. The response surfaces' steepness demonstrated how variables affected responses. More impact of variables on response was indicated by the steeper surfaces. Moreover, the density of contour lines displayed how various factors affected responses. The peak value in\u003c/em\u003e Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e \u003cem\u003e(d, e, f, g, h, and i) represented the perfect value for nanoliposome count and EE. In contrast, the valley value in\u003c/em\u003e Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e \u003cem\u003e(a, b, and c) represented the ideal value for the size of produced liposomes. The outline's shape offers additional proof of how two factors interacted with one another. Furthermore, the contours' shape (Fig. S2) also demonstrated the strong and weak interactions between the two factors. The close circle showed that there was no significant interaction between the two factors\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e: Estimated response surface for (a,d,g) pressure drop was fixed at 22.5 MPa, (b,f,h) flow rate was fixed at 2 mL/min, (c,e,i) temperature was fixed at 52.5 ˚C for the mean size, Count, and EE, respectively.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\n\u003ch2\u003e3.1.6. Optimization condition\u003c/h2\u003e\n\u003cp\u003eThe resulting model was used to forecast the ideal conditions for producing irinotecan PEGylated liposomes by the ESSAS method. The ideal conditions for the produced nanoliposomes included a pressure drop at 29.9 MPa, a flow rate at 2.4 mL/min, and a temperature oat 42\u0026deg;C to maximize the EE and count of created liposomes while minimizing the size. The predicted mean size, count, and EE of the produced liposomes could reach 58 nm, 663, and 91.5% under optimization conditions. The test was run under the aforementioned circumstances three times to evaluate the anticipated model's adaptability and optimal points with an experimental test. As seen in Fig.\u0026nbsp;5a, 758 nanoliposomes with an average size of 55 nm were created. Figure\u0026nbsp;5b also shows the size distribution of liposomes produced at the optimal point. The EE for produced nanoliposomes also reached 94.6% in this test, confirming the remarkable agreement between the projected model and the experimental test under ideal conditions.\u003c/p\u003e\n\u003cp\u003eCompared to other studies illustrated in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, it is obvious that the liposomes created in this work had encapsulation efficiency equal to or greater than studies that produced irinotecan liposomes. In contrast, the size of the liposomes generated in this work was significantly smaller than those. Due to the smaller liposomes produced, we expect them to be less recognized by macrophages and remain in the circulatory system longer. Additionally, only safe solvents, water, and ethanol have been used in this study. In contrast, harmful organic solvents have been employed in other studies.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eComparing the results of this work with other article which produced irinotecan liposome\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eArticles\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMean size (nm)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eEE (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSolvent and Chemical component\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e116\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e98\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEthanol/ Triethylamine/ Sodium sucrose octasulfate\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e198\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e91\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAmmonium sulfate/ Citric acid/ Dehydrated alcohol/ NaOH\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e69\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e94\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOctadecylamine/ Chloroform/ Ammonium Sulfate\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e150\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e~\u0026thinsp;90\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEthanol/ Triethanolamine/ CuSO\u003csub\u003e4\u003c/sub\u003e/ H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e/ Citrate buffer/ EDTA/ Sucrose/ HEPES\u0026nbsp;(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eThis work\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e55\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e95\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEthanol/Water\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003ch2\u003e3.2. Storage stability study\u003c/h2\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe storage stability of the IRH PEGylated liposomes made under ideal conditions was examined over 8 weeks. To assess the impact of time on the stability of the manufactured liposomes, they were stored at 4\u0026deg;C in the dark. At various time intervals, the mean diameter of liposomes and their EE were examined (Fig.\u0026nbsp;5d). As seen in Fig. S4d, the initial EE and particle size for PEGylated liposomes were found to be 93.8% and 55 nm, respectively. After 2 months, the EE reduced to 91.3% while the mean size of IRH PEGylated liposomes reached 61 nm. The experiment showed minimal change in initial IRH PEGylated liposomes kept at 4\u0026deg;C and approved these liposomes were stable for up to 60 days with no discernible changes in size and EE. This occurrence happened because liposomes were thermodynamically stable due to the homogeneous charge distribution on their surface. However, a slight change in liposome size resulted from their breakdown. Furthermore, even after storage, the spherical liposomes were still visible in the FESEM image (Fig.\u0026nbsp;5c). These results also demonstrated the high stability of the produced IRH liposomes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003e3.3. Irinotecan hydrochloride release test\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eTo determine how liposomes affected the kinetics of drug release, drug release experiments were carried out at 37 C. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e displays the release profile of IRH from PEGylated and conventional liposomes and the Free Drug as a Control. According to the results, drug penetration through the dialysis membrane was not a limiting step because the release of free IRH was rapid, and 97% of the drug was released over 5 hours. On the other hand, during the initial sampling period (120 min), it was discovered that both PEGylated and conventional liposomes had an initial burst in the drug release that was greater than 18% and 10% for non-pegylated and pegylated liposomes, respectively. The observed burst release may be due to the release of the IRH that was adsorbed on the surface of the liposome. A steady drug release was observed following the first burst release, and over 10 hours, about 22 and 46% of the drug from PEGylated and traditional liposomes were released. The dissolved medication within the liposome's core diffusing into the dissolution medium may cause this delayed release. Moreover, the release rate of irinotecan from PEGylated liposomes was much lower than that from non-PEGylated Liposomes because of the more rigid bilayers of PEGylated Liposomes.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis work used the ESSAS method to generate PEGylated liposomes of the powerful anticancer medication irinotecan hydrochloride. The Box-Behnken design was used to analyze how temperature, pressure drop, and flow rate affected the properties of the IRH liposomes and identify the optimum condition. It was discovered that the model developed in this study could accurately predict the experimental data. Furthermore, it was found that the pressure drop of 29.9 MPa, temperature of 42\u0026deg;C, and flow rate of 2.4 mL/min were the ideal conditions for the production of IRH PEGylated liposomes. Under ideal circumstances, liposomes with EE\u0026thinsp;=\u0026thinsp;94.6%, count\u0026thinsp;=\u0026thinsp;758, and dimension\u0026thinsp;=\u0026thinsp;55 nm were created. Besides that, the flow rate had a significant impact on the EE, and the pressure drop was the factor that had the biggest impact on the count and size of the PEGylated liposomes. These findings showed that, in contrast to most traditional methods, this technique produced smaller IRH liposomes with high EE and without harmful solvents. Furthermore, the ESSAS process requires one step and does not require further extrusion or ultrasonication phases. As a result, this technology is a quick and easy substitute for conventional methods to produce liposomes with the best and most controllable physicochemical features (size) while also being able to be applied in large-scale industrial applications and adaptable to an industrial GMP process. In the future, liposomes containing both hydrophobic and hydrophilic active compounds will be produced using this technique. Furthermore, modified coatings of liposomes will be investigated to create targeted-release vesicles.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.M.: Methodology, doing experiment, writing original draft. M.K.: design, editing text, interpret the data. F.R.: Supervise the project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by Shahid Beheshti University (Grant No.1398).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agreed with the content and give their consent to submit the manuscript for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAll authors: Department of Analytical Chemistry and pollutants, Shahid Beheshti University, Tehran, Iran\u003c/em\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYang T, Choi M-K, Cui F-D, Kim J S, Chung S-J, Shim C-K,Kim D-D. Preparation and evaluation of paclitaxel-loaded PEGylated immunoliposome. JCR. 2007;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jconrel.2007.05.011\u003c/span\u003e\u003cspan address=\"10.1016/j.jconrel.2007.05.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi S, Kang B, Yang E, Kim K, Kwak M K, Chang P-S,Jung H-S. Precise control of liposome size using characteristic time depends on solvent type and membrane properties. Scientific Reports 2023;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-023-31895-z\u003c/span\u003e\u003cspan address=\"10.1038/s41598-023-31895-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrucillo P, Campardelli R, Scognamiglio M,Reverchon E. Control of liposomes diameter at micrometric and nanometric level using a supercritical assisted technique. J. CO2 Util. 2019;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jcou.2019.04.014\u003c/span\u003e\u003cspan address=\"10.1016/j.jcou.2019.04.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu D, Cohen J,Turkman N. PEG2000-DBCO surface coating increases intracellular uptake of liposomes by breast cancer xenografts. Sci. Rep. 2022; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-022-14947-8\u003c/span\u003e\u003cspan address=\"10.1038/s41598-022-14947-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAtyabi F, Farkhondehfai A,Esmaeili F. Preparation of pegylated nano-liposomal formulation containing SN-38: in vitro characterization and in vivo biodistribution in mice. Acta pharmaceutica 2009;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003eHttps://doi.org/10.2478/v10007-009-0020-0\u003c/span\u003e\u003cspan address=\"Https://10.2478/v10007-009-0020-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDadashzadeh S, Mirahmadi N, Babaei M H,Vali A M. Peritoneal retention of liposomes: Effects of lipid composition, PEG coating and liposome charge. JCR. 2010;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jconrel.2010.08.026\u003c/span\u003e\u003cspan address=\"10.1016/j.jconrel.2010.08.026\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakamura K, Yamashita K, Itoh Y, Yoshino K, Nozawa S,Kasukawa H. Comparative studies of polyethylene glycol-modified liposomes prepared using different PEG-modification methods. Biochim. Biophys. Acta - Biomembr. 2012;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbamem.2012.06.019\u003c/span\u003e\u003cspan address=\"10.1016/j.bbamem.2012.06.019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSyama K, Jakubek Z J, Chen S, Zaifman J, Tam Y Y C,Zou S. Development of lipid nanoparticles and liposomes reference materials (II): cytotoxic profiles. Sci. Rep. 2022;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-022-23013-2\u003c/span\u003e\u003cspan address=\"10.1038/s41598-022-23013-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMirhadi E \u003cem\u003eet al.\u003c/em\u003e Redox-sensitive doxorubicin liposome: a formulation approach for targeted tumor therapy. Sci. Rep. 2022;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-022-15239-x\u003c/span\u003e\u003cspan address=\"10.1038/s41598-022-15239-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVali A M, Toliyat T, Shafaghi B,Dadashzadeh S. Preparation, Optimization, and Characterization of Topotecan Loaded PEGylated Liposomes Using Factorial Design. Drug Dev. Ind. Pharm. 2008; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/03639040701385055\u003c/span\u003e\u003cspan address=\"10.1080/03639040701385055\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHashemzadeh H, Javadi H,Darvishi M H. Study of Structural stability and formation mechanisms in DSPC and DPSM liposomes: A coarse-grained molecular dynamics simulation. Sci. Rep. 2020;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-020-58730-z\u003c/span\u003e\u003cspan address=\"10.1038/s41598-020-58730-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarrion C, Domingo J C,de Madariaga M A. Preparation of long-circulating immunoliposomes using PEG\u0026ndash;cholesterol conjugates: effect of the spacer arm between PEG and cholesterol on liposomal characteristics. Chem. Phys. Lipids. 2001;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0009-3084(01)00178-5\u003c/span\u003e\u003cspan address=\"10.1016/S0009-3084(01)00178-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang J \u003cem\u003eet al.\u003c/em\u003e The preparation, characterization of Lupeol PEGylated liposome and its functional evaluation in vitro as well as pharmacokinetics in rats. Drug Dev. Ind. Pharm. 2019;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/03639045.2019.1569038\u003c/span\u003e\u003cspan address=\"10.1080/03639045.2019.1569038\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei H \u003cem\u003eet al.\u003c/em\u003e Active loading liposomal irinotecan hydrochloride: Preparation, in vitro and in vivo evaluation. Asian Journal of Pharmaceutical Sciences 2013;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ajps.2013.10.006\u003c/span\u003e\u003cspan address=\"10.1016/j.ajps.2013.10.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiao H,Sedlař\u0026iacute;k V. A Rapid and Sensitive HPLC Method for Simultaneous Determination of Irinotecan Hydrochloride and Curcumin in Co-delivered Polymeric Nanoparticles. Journal of Chromatographic Science 2020;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/chromsci/bmaa033\u003c/span\u003e\u003cspan address=\"10.1093/chromsci/bmaa033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRajpoot K,Jain S K. Irinotecan hydrochloride trihydrate loaded folic acid-tailored solid lipid nanoparticles for targeting colorectal cancer: development, characterization, and in vitro cytotoxicity study using HT-29 cells. Journal of Microencapsulation 2019;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/02652048.2019.1665723\u003c/span\u003e\u003cspan address=\"10.1080/02652048.2019.1665723\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoudel B K \u003cem\u003eet al.\u003c/em\u003e Development of polymeric irinotecan nanoparticles using a novel lactone preservation strategy. Int. J. Pharm. 2016;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijpharm.2016.08.018\u003c/span\u003e\u003cspan address=\"10.1016/j.ijpharm.2016.08.018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y \u003cem\u003eet al.\u003c/em\u003e Pharmacovigilance of herb-drug interactions: A pharmacokinetic study on the combination administration of herbal Kang\u0026rsquo;ai injection and chemotherapy irinotecan hydrochloride injection by LC\u0026ndash;MS/MS. Journal of Pharmaceutical and Biomedical Analysis 2021;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jpba.2020.113784\u003c/span\u003e\u003cspan address=\"10.1016/j.jpba.2020.113784\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNaik S, Patel D, Surti N,Misra A. Preparation of PEGylated liposomes of docetaxel using supercritical fluid technology. J. Supercrit. Fluids. 2010;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.supflu.2010.02.005\u003c/span\u003e\u003cspan address=\"10.1016/j.supflu.2010.02.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrucillo P,Reverchon E. Production of PEG-coated liposomes using a continuous supercritical assisted process. The Journal of Supercritical Fluids 2021;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.supflu.2020.105048\u003c/span\u003e\u003cspan address=\"10.1016/j.supflu.2020.105048\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePenoy N, Grignard B, Evrard B,Piel G. A supercritical fluid technology for liposome production and comparison with the film hydration method. Int. J. Pharm. 2021;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijpharm.2020.120093\u003c/span\u003e\u003cspan address=\"10.1016/j.ijpharm.2020.120093\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarbi I, Aljaeid B, El-Say K M,Zidan A S. Glycosylated Sertraline-Loaded Liposomes for Brain Targeting: QbD Study of Formulation Variabilities and Brain Transport. AAPS PharmSciTech 2016;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1208/s12249-016-0481-7\u003c/span\u003e\u003cspan address=\"10.1208/s12249-016-0481-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhangari H, King J W, Ehsani A,Yousefi M. Supercritical fluid extraction of seed oils \u0026ndash; A short review of current trends. Trends Food Sci. Technol. 2021;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tifs.2021.02.066\u003c/span\u003e\u003cspan address=\"10.1016/j.tifs.2021.02.066\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChauhan T \u003cem\u003eet al.\u003c/em\u003e Negatively charged liposomes of sertraline hydrochloride: Formulation, characterization and pharmacokinetic studies. Journal of Drug Delivery Science and Technology 2020;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jddst.2020.101780\u003c/span\u003e\u003cspan address=\"10.1016/j.jddst.2020.101780\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaja L, Željko K,Mateja P. Sustainable technologies for liposome preparation. The Journal of Supercritical Fluids 2020;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.supflu.2020.104984\u003c/span\u003e\u003cspan address=\"10.1016/j.supflu.2020.104984\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalehi H, Karimi M,Raofie F. Micronization and coating of bioflavonoids extracted from Citrus sinensis L. peels to preparation of sustained release pellets using supercritical technique. JICS. 2021;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s13738-021-02262-4\u003c/span\u003e\u003cspan address=\"10.1007/s13738-021-02262-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanto I E, Campardelli R, Albuquerque E C, de Melo S V, Della Porta G,Reverchon E. Liposomes preparation using a supercritical fluid assisted continuous process. Chemical Engineering Journal 2014;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cej.2014.03.099\u003c/span\u003e\u003cspan address=\"10.1016/j.cej.2014.03.099\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarimi M, Raofie F,Karimi M. Production Ganoderma lucidum extract nanoparticles by expansion of supercritical fluid solution and evaluation of the antioxidant ability. Scientific Reports 2022;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-022-13727-8\u003c/span\u003e\u003cspan address=\"10.1038/s41598-022-13727-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharifi F, Zhou R, Lim C, Jash A, Abbaspourrad A,Rizvi S S H. Generation of liposomes using a supercritical carbon dioxide eductor vacuum system: Optimization of process variables. Journal of CO2 Utilization 2019;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jcou.2018.12.011\u003c/span\u003e\u003cspan address=\"10.1016/j.jcou.2018.12.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohammadi M, Karimi M,Raofie F. Expansion supercritical fluid into an aqueous solution (ESSAS), a new technique for creating nano-size cyanocobalamin-loaded liposomes, and optimization of involved parameters. JICS. 2023;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s13738-023-02930-7\u003c/span\u003e\u003cspan address=\"10.1007/s13738-023-02930-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYousefi M, Rahimi-Nasrabadi M, Pourmortazavi S M, Wysokowski M, Jesionowski T, Ehrlich H,Mirsadeghi S. Supercritical fluid extraction of essential oils. TrAC Trends in Analytical Chemistry 2019;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.trac.2019.05.038\u003c/span\u003e\u003cspan address=\"10.1016/j.trac.2019.05.038\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTavares Luiz M \u003cem\u003eet al.\u003c/em\u003e Design of experiments (DoE) to develop and to optimize nanoparticles as drug delivery systems. European Journal of Pharmaceutics and Biopharmaceutics 2021;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ejpb.2021.05.011\u003c/span\u003e\u003cspan address=\"10.1016/j.ejpb.2021.05.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJacyna J, Kordalewska M,Markuszewski M J. Design of Experiments in metabolomics-related studies: An overview. Journal of Pharmaceutical and Biomedical Analysis 2019;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jpba.2018.11.027\u003c/span\u003e\u003cspan address=\"10.1016/j.jpba.2018.11.027\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAslan N,Cebeci Y. Application of Box\u0026ndash;Behnken design and response surface methodology for modeling of some Turkish coals. Fuel. 2007;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fuel.2006.06.010\u003c/span\u003e\u003cspan address=\"10.1016/j.fuel.2006.06.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBezerra M A, Santelli R E, Oliveira E P, Villar L S,Escaleira L A. Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta 2008;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.talanta.2008.05.019\u003c/span\u003e\u003cspan address=\"10.1016/j.talanta.2008.05.019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalehi H, Karimi M, Rezaie N,Raofie F. Extraction of β-Carboline alkaloids and preparation of extract nanoparticles from Peganum harmala L. capsules using supercritical fluid technique. Journal of Drug Delivery Science and Technology 2020;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jddst.2020.101515\u003c/span\u003e\u003cspan address=\"10.1016/j.jddst.2020.101515\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVarona S, Mart\u0026iacute;n \u0026Aacute;,Cocero M J. Liposomal Incorporation of Lavandin Essential Oil by a Thin-Film Hydration Method and by Particles from Gas-Saturated Solutions. Industrial \u0026amp; Engineering Chemistry Research 2011;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/ie102016r\u003c/span\u003e\u003cspan address=\"10.1021/ie102016r\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL\u0026eacute;vai G, Mart\u0026iacute;n \u0026Aacute;, de Paz E, Rodr\u0026iacute;guez-Rojo S,Cocero M J. Production of stabilized quercetin aqueous suspensions by supercritical fluid extraction of emulsions. J. Supercrit. Fluids. 2015;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.supflu.2015.02.019\u003c/span\u003e\u003cspan address=\"10.1016/j.supflu.2015.02.019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu S, Yang F, Zhang C, Ji H, Hong P,Deng C. Optimization of process parameters for supercritical carbon dioxide extraction of Passiflora seed oil by response surface methodology. J. Supercrit. Fluids. 2009;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.supflu.2008.09.013\u003c/span\u003e\u003cspan address=\"10.1016/j.supflu.2008.09.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiao Z, Wang X, Han S, Zha X,Xia J. Preparation of vitamin C liposomes by rapid expansion of supercritical solution process: Experiments and optimization. Journal of Drug Delivery Science and Technology 2019;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jddst.2019.02.015\u003c/span\u003e\u003cspan address=\"10.1016/j.jddst.2019.02.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang T, He W, Du Y, Wang J,Li X. Redox-sensitive irinotecan liposomes with active ultra-high loading and enhanced intracellular drug release. Colloids and Surfaces B: Biointerfaces 2021;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.colsurfb.2021.111967\u003c/span\u003e\u003cspan address=\"10.1016/j.colsurfb.2021.111967\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Z,Yao J. Preparation of Irinotecan-Loaded Folate-Targeted Liposome for Tumor Targeting Delivery and Its Antitumor Activity. AAPS PharmSciTech 2012;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1208/s12249-012-9776-5\u003c/span\u003e\u003cspan address=\"10.1208/s12249-012-9776-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang L, Cao D Y, Wang J, Xiang B, Dun J N, Fang Y,Xue G Q. PEG-coated irinotecan cationic liposomes improve the therapeutic efficacy of breast cancer in animals. Eur Rev Med Pharmacol Sci 2013;\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHattori Y, Shi L, Ding W, Koga K, Kawano K, Hakoshima M,Maitani Y. Novel irinotecan-loaded liposome using phytic acid with high therapeutic efficacy for colon tumors. J Control Release 2009;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jconrel.2009.01.013\u003c/span\u003e\u003cspan address=\"10.1016/j.jconrel.2009.01.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":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":"discover-nano","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"narl","sideBox":"Learn more about [Discover Nano](https://www.springer.com/journal/11671)","snPcode":"11671","submissionUrl":"https://submission.nature.com/new-submission/11671/3","title":"Discover Nano","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Drug delivery systems, Supercritical fluid expansion, Liposomes, Irinotecan, Polyethylene glycol, Response surface methodolog","lastPublishedDoi":"10.21203/rs.3.rs-4492900/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4492900/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA semi-synthetic camptothecin derivative known as irinotecan hydrochloride is frequently used to treat colorectal cancer (CRC), including colorectal adenocarcinoma and lung cancers involving small cells. Irinotecan has a very short half-life; therefore, continuous infusions are required to keep the drug's blood levels at therapeutic levels, which could produce cumulative toxicities. Effective delivery techniques, including liposomes, have been developed to address these shortcomings. In this study, a continuous supercritical fluid approach dubbed Expansion Supercritical Fluid into an aqueous solution (ESSAS), in which the pressure decreases rapidly but remains over the critical pressure, is proposed to manufacture polyethylene glycolylated (PEGylated) liposomes carrying irinotecan hydrochloride. To accomplish this, PEGylated liposomes were created using a Box-Behnken design, and the operating parameters (flow rate, temperature, and pressure drop) were optimized. Encapsulation efficiency, mean size, and prepared liposome count were 94.6%, 55 nm, and 758 under ideal circumstances. Additionally, the stability of the PEGylated liposome was investigated during 8 weeks, and also PEGylated liposome-loaded irinotecan release profile was compared to conventional liposomes and free irinotecan, and a constant drug release was seen after the first burst release from liposomes.\u003c/p\u003e","manuscriptTitle":"Preparation Irinotecan Hydrochloride Loaded PEGylated Liposomes Using Novel Method Supercritical Fluid and Condition Optimized by Box-Behnken Design","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-14 07:40:28","doi":"10.21203/rs.3.rs-4492900/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-13T11:26:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-10T01:34:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"42626485314823562357854167905076328660","date":"2024-06-04T11:39:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-04T08:44:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"267402080942165440782693932360795298245","date":"2024-06-04T05:51:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-04T05:32:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-03T11:11:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-03T11:10:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Nano","date":"2024-05-28T19:39:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-nano","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"narl","sideBox":"Learn more about [Discover Nano](https://www.springer.com/journal/11671)","snPcode":"11671","submissionUrl":"https://submission.nature.com/new-submission/11671/3","title":"Discover Nano","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"add06cf5-d8ab-4af5-8c64-6819334dc032","owner":[],"postedDate":"June 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-09T16:18:06+00:00","versionOfRecord":{"articleIdentity":"rs-4492900","link":"https://doi.org/10.1186/s11671-024-04071-z","journal":{"identity":"discover-nano","isVorOnly":false,"title":"Discover Nano"},"publishedOn":"2024-09-05 16:08:24","publishedOnDateReadable":"September 5th, 2024"},"versionCreatedAt":"2024-06-14 07:40:28","video":"","vorDoi":"10.1186/s11671-024-04071-z","vorDoiUrl":"https://doi.org/10.1186/s11671-024-04071-z","workflowStages":[]},"version":"v1","identity":"rs-4492900","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4492900","identity":"rs-4492900","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}