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 [7,8,9].

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, CO2 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) [27,28,29,30].

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 (mPEG1500-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 min 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 cm3 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.

Fig. 1
figure 1

The 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

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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 min, 40 MPa, and 30 min, 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 min, 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 min 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 CO2 (SC-CO2) was passed from it. At this stage, a sudden pressure drop occurred. As a result, the density and solubility of SC-CO2 decreased, and using nucleation, substances dissolved in SC-CO2 began to precipitate in the production vessel. Eventually, during this procedure, the precipitated phospholipids, mPEG1500-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 min. 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-min analysis at 1 mL/min and 379 nm, respectively. Irinotecan hydrochloride standard curves were created using the drug standard at 10–500 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.

$$\text{EE\% }=\frac{\text{Ct }-\text{ F}}{\text{Ct }} \times 100\text{\%}$$
(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-CO2. The pressure was then quickly reduced during the production step by opening valves 2 and 3, allowing SC-CO2 to flow into vessel 2. This caused the creation of fine water droplets and reduced the density and solvent power of SC-CO2. As a result, phospholipid, cholesterol, and mPEG1500-DSPE were precipitated from SC-CO2 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 mPEG1500-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 min, 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 min. 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 and p > 0.05 denote significant and insignificant results, respectively.

Table 1 Design matrix and the responses (BBD) for the production of irinotecan PEGylated nanoliposomes
Full size table

The formulas of the second-order polynomial equations resulting from the experimental designs were obtained as follows:

$$\begin{aligned} {\text{Y}}_{{1}} & = {588}.{895} - {42}.{125}\,{\text{X}}_{{1}} - {13}.{2333}\,{\text{X}}_{{2}} - {11}.{4}0{6}\,{\text{X}}_{{3}} + {3}.0\,{\text{X}}_{{1}}^{{2}} + 0.{366667}\,{\text{X}}_{{1}} {\text{X}}_{{2}} \\ & \quad + 0.{4}\,{\text{X}}_{{1}} {\text{X}}_{{3}} + 0.{226667}\,{\text{X}}_{{2}}^{{2}} - 0.0{133333}\,{\text{X}}_{{2}} {\text{X}}_{{3}} + 0.0{992}\,{\text{X}}_{{3}}^{{2}} \\ \end{aligned}$$
(2)
$$\begin{aligned} {\text{Y}}_{{2}} & = - {3542}.{36} + {689}.{717}\,{\text{X}}_{{1}} + {84}.{3167}\,{\text{X}}_{{2}} + {8}0.{714}\,{\text{X}}_{{3}} - {112}.{417}\,{\text{X}}_{{1}}^{{2}} - {1}.{9}\,{\text{X}}_{{2}} \\ & \quad - {3}.{82}\,{\text{X}}_{{1}} {\text{X}}_{{3}} + 0.{228148}\,{\text{X}}_{{2}}^{{2}} - {1}.{22667}\,{\text{X}}_{{2}} {\text{X}}_{{3}} - 0.{487467}\,{\text{X}}_{{3}}^{{2}} \\ \end{aligned}$$
(3)
$$\begin{aligned} {\text{Y}}_{{3}} & = - {45}.{58} + {2}.{95}\,{\text{X}}_{{1}} + {2}.{78}\,{\text{X}}_{{2}} + {3}.{564}\,{\text{X}}_{{3}} - {3}.{625}\,{\text{X}}_{{1}}^{{2}} + 0.{766667}\,{\text{X}}_{{1}} {\text{X}}_{{2}} \\ & \quad + 0.0{2}\,{\text{X}}_{{1}} {\text{X}}_{{3}} - 0.0{644444}\,{\text{X}}_{{2}}^{{2}} - 0.0{186667}\,{\text{X}}_{{2}} {\text{X}}_{{3}} - 0.0{328}\,{\text{X}}_{{3}}^{{2}} \\ \end{aligned}$$
(4)

where Y1, Y2, and Y3 stand for the mean size, count of PEGylated liposomes, and EE. Furthermore, the X1 through X3 stands for the flow rate (mL/min), pressure drop (MPa), and temperature (°C), respectively.

The ANOVA results showed good R2 and R2 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. 2d–f) when one parameter is altered while the others are kept constant. Additionally, the Pareto chart (Fig. 2a–c) shows the parameters’ impact and specifies which factors significantly impacted the responses at a 95% confidence level.

Fig. 2
figure 2

Standardized 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

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Figure 2d 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-CO2 [19]. As a result, it created a greater difference between the value of phospholipids that could be dissolved in SC-CO2 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. 2e, the count of produced liposomes increased.

As indicated in Fig. 2f, 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 3a–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. 2a–c) demonstrates that pressure drop significantly impacts the mean size, count, and EE of the production process.

Fig. 3
figure 3

FESEM 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

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3.1.3 Temperature effect

This study looked into the effects of temperatures from 40 to 65 °C. According to Fig. 2a–c the mean size, count, and EE of liposomes were significantly influenced by temperature.

According to Fig. 2d–f, the temperature had a reverse effect in this study. Firstly, the rising temperature caused more phospholipid soluble in CO2 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-CO2 decreased. As a result, it decreased the solubility of the compounds and the solvent capacity of SC-CO2, 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–h, 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 1–3 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-CO2 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. 2d–f, increasing the fluid flow rate resulted in higher mixing energy and greater atomization of cargo solution droplets in SC-CO2 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. Figure 3i–l, 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. 4d–i represented the perfect value for nanoliposome count and EE. In contrast, the valley value in Fig. 4a–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, whereas the ellipse showed that there was significant interaction.

Fig. 4
figure 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

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Figure 4c shows that how the size of liposomes altered at a mean level of temperature of 55 °C when flow rate and pressure drop increased. Moreover, Fig. 4a demonstrated that rising flow rate and temperature at a constant pressure drop would slightly impact this response. Additionally, when temperature and pressure drop rise, smaller liposomes (42 nm) are formed, and this interaction has a significant impact (Fig. 4b). Equation (2) demonstrated that the X1X2 and X1X3 interaction coefficients were positive while the X2X3 was negative. Additionally, as shown in Fig. 4d–h, the number of liposomes and EE that were produced rose to a certain level with the development in flow rate and temperature as well as the pressure drop and temperature, and then it started to fall. Also, these responses increased with increasing pressure drop and flow rate, as seen in Fig. 4f, i. Additionally, as shown in Eq. (3), all of the interaction coefficients for the count of PEGylated liposomes were negative. However, for the EE, the interactions between X1X2 and X1X3 were positive, and X2X3 had a negative effect.

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 ± 22.7 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% ± 1.7 in this test, confirming the remarkable agreement between the projected model and the experimental test under ideal conditions.

Fig. 5
figure 5

a 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

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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
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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 h. 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 h, 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.

Fig. 6
figure 6

Drug release test of irinotecan hydrochloride from PEGylated liposomal formulation, traditional liposomes, and pure irinotecan hydrochloride solution as control at different periods of time

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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% ± 1.7, count = 758, and dimension = 55 ± 22.7 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.