Propofol-loaded nanomicelle with improved anesthetic, pharmacokinetic, hemocompatibility, safety, and permeation profiles

2.1 Materials

Propofol, carboxylic acid-poly [ethylene glycol (COO-PEG)]-b-poly [ D, L lactide (PDLA)], PEG average M_n 5000, PDLA average M_n 16,000 were purchased from Sigma Company (St Louis, USA). All chemicals were analytical grade and distilled water was employed in all assays.

2.2 Preparation of propofol-COO-PEG-PLDA nanomicelle

Propofol (5 mg) and COO-PEG-PLDA (30 mg) were hydrated in methanol (10 mL), followed by addition of distilled water dropwise to the solution, incubation at 50 °C for 10 min, vortex for 10 s, filtered (0.22 µm) and finally freeze-dried. Afterward, 5 mg of propofol-COO-PEG-PLDA nanomicelle was rehydrated in distilled water and the propofol loading capacity and nanoparticle yield was calculated based on the reported equations in the previous reports (Wang et al., 2017; Li et al., 2017) using UV/VIS absorbance of at 285 nm (Shimadzu UV-1900i, Japan).

2.3 Characterization of propofol-COO-PEG-PLDA nanomicelle

The characterization of propofol-COO-PEG-PLDA nanomicelle was done by transmission electron microscope (TEM) (Zeiss-EM10C-100 KV, Germany) and dynamic light scattering (DLS) (ZEN314, England) analysis. Following lyophilization, propofol-COO-PEG-PLDA nanomicelle was rehydrated at 0.1 mg/mL distilled water, centrifuged (2500 rpm) and analyzed.

2.4 Drug release assay

The propofol release assay was done by a UV/VIS absorbance of at 285 nm (Shimadzu UV-1900i, Japan) using two different pH: 7.4 and 4.5, which represent the pH of blood and lysosomes and endosomes (Kievit et al., 2011), respectively. 2 mL of propofol-COO-PEG-PLDA nanomicelle solution poured into dialysis bags (MWCO 3.5 kDa) and placed in 48 mL of release medium at 37 °C for 120 min. At different time intervals, 0.5 mL of release medium was withdrawn and propofol concentration was detected at 285 nm.

2.5 Cytotoxic effect against human peripheral blood mononuclear cells (PBMCs)

This assay was done to assess the effect of the propofol in the form if free or nano-formulated state on normal human cells. To isolate the PBMCs, fresh blood samples (10 mL) were collected under medical and ethical committee control from healthy volunteer donors and the cells were isolated through Ficoll-hypaque density centrifugation method. The cytotoxic effect was then evaluated by the MTT assay.

2.6 MTT assay

After incubation of cells with serial concentrations of propofol or propofol-COO-PEG-PLDA nanomicelle (1, 2, 5, 10, 20, 50, 100, and 200 μg/mL) for 24 h at 37 °C and 5% CO₂, a fixed volume of medium was gently removed and replaced by 20 μL of MTT solution (5 mg/mL of PBS) and incubated for 4 h. The plates were then treated with 200 μL of DMSO for 5 min at 37°Cfolowed by reading the absorbance of samples at 540 nm using a Multiskan MK3 microplate reader (Labsystems, Helsinki, Finland).

2.7 Hemolytic assay

Fresh human red blood cells (RBCs) were centrifuged (3000 rpm, 10 min) at 4 °C, washed, diluted 12-fold with PBS, added to a 200 μL of propofol or propofol-COO-PEG-PLDA nanomicelle in PBS (pH 7.4) at varying concentrations of 1, 2, 5, 10, 20, 50, 100, and 200 μg/mL. PBS and distilled water were used as negative and positive controls, respectively. The solutions were vortexed, incubated at 37 °C for 2 h, and centrifuged (3000 rpm, 5 min). A total of 100 μL of supernatant was then removed and the absorbance values of the supernatants were read at 540 nm employing a Multiskan MK3 microplate reader (Labsystems, Helsinki, Finland). The zero hemolysis (0%) and the total hemolysis (100%) was assigned when the samples were in the presence of PBS and distilled water, respectively.

2.8 Permeation assay

In vitro permeation of 1% propofol solution or 1% propofol-COO-PEG-PLDA nanomicelle across rat esophagus epithelium was caried out in Franz type vertical diffusion cells with permeation area of 1.98 cm² and receptor compartment of 10 mL in volume under constant magnetic stirring (300 rpm) at 37 °C for 5 h. The epithelium was placed between chambers over a cellulose filter without any damage to the barrier. The compartments were filled with filtered/degassed PBS (pH 7.4) and the donor chamber buffer was replaced by 1 mL of either % propofol solution or 1% propofol-COO-PEG-PLDA nanomicelle. The samples were analyzed by HPLC and the steady-state flux (J_ss) and lag time (t_lag) were obtained by the linear portion of the cumulative amount of propofol permeated per cm² of esophagus epithelium (Q) over time.

2.9 Pharmacokinetic assay in vivo

Female Sprague-Dawley rats (250 g) were randomly divided into four groups and were given a single injection of free propofol (150 mg/kg, over 5 min, 0.4 mL) or propofol-COO-PEG-PLDA nanomicelle (150 mg/kg propofol-equivalent dosing dissolved in 150 mM NaCl (n = 10). Two control groups received 150 mM NaCl and equivalent dose of COO-PEG-PLDA nanomicelle (n = 8). About 0.25 mL of blood was collected through the catheter after different time intervals (0,2, 5, 10, 20, 30, 50, 100, and 120 min), centrifuged (2000 rpm, 10 min), diluted plasma 1: 4 in cold 0.1% phosphoric acid in methanol, vortexed, centrifuged (13,000 rpm,10 min), and analyzed by HPLC. Animals were handled in accordance with ethical rules approved by Department of Anesthesiology, Zhengzhou University, China.

2.10 Hind paw model

After pacing the rats in wired mesh floor cage 45 min, enhancing forces were applied to the wound hyperalgesic based on previous study (Grant et al., 1997). Hyperalgesic response was defined based on the paw withdrawal with a force 20% of baseline. Afterwards, wounds were infiltrated with 0.3 mL propofol 1%, propofol-COO-PEG-PLDA nanomicelle (n = 10) 1% or controls (NaCl or nanomicelle, n = 8). The rats were then exposed to the forces and the absence of reflex (paw withdrawal) was considered as anesthesia (Grant et al., 1997). In this study, the time interval between drug injection and the balance reflex dilution was recorded as the induction time of anesthesia. The interval between the loss of the balance reflex and its return was the length of the anesthesia period. The interval between the disappearance of the balance reflex and the animal's ability to walk again after anesthesia was defined as the walking time.

2.11 Statistical analysis

All data are expressed as mean ± standard deviation (SD) of three independent assays. Statistical analysis of significance was reported based on Student’s t test. P < 0.05 is considered as statistically significant.

3.1 Characterization of propofol-COO-PEG-PLDA nanomicelle

The TEM photo of propofol-COO-PEG-PLDA nanomicelle is depicted in Fig. 1A. They show a smooth spherical morphology with a diameter of about 20–30 nm. To explore the colloidal stability, DLS study was run to determine the hydrodynamic radius, polydispersity index (PDI) and zeta potential values of fabricated propofol-COO-PEG-PLDA nanomicelle. Results shown in Fig. 1B indicated that the particles of COO-PEG-PLDA nanomicelle and propofol-COO-PEG-PLDA nanomicelle are 63.29 nm (PDI: 0.193) and 73.24 nm (PDI: 0.201), respectively. Also, zeta potential values indicated that the charge distribution of COO-PEG-PLDA nanomicelle and propofol-COO-PEG-PLDA nanomicelle samples are −19.67 mV and –23.59 mV, respectively (Fig. 1C). The data indicated that loading of nanomicelles by propofol slightly increases the hydrodynamics radius and zeta potential values of complex, pointing out the potential loading of drug and colloidal stability of the nano-based system.

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Fig. 1

(A) TEM image of propofol-COO-PEG-PLDA nanomicelle, (B) hydrodynamic radius analysis of COO-PEG-PLDA nanomicelle (blue) and propofol-COO-PEG-PLDA nanomicelle (red), (C) zeta potential value of COO-PEG-PLDA nanomicelle (blue) and propofol-COO-PEG-PLDA nanomicelle (red), (D) hydrodynamic radius of propofol-COO-PEG-PLDA nanomicelle over time (120 min). Data are reported as mean ± SD (n = 3).

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Also, to explore the stability of propofol-COO-PEG-PLDA nanomicelle, DLS study was run for 120 min and during different time intervals, hydrodynamic radius was determined. As shown in Fig. 1D, the hydrodynamic radius of propofol-COO-PEG-PLDA nanomicelle is almost constant for 50 min, afterward it sharply increases, implying that the structure integrity of nanocomplex is stable during 50 min.

3.2 Drug release assay

It was found that the encapsulation efficiency and the drug loading capacity of propofol-COO-PEG-PLDA nanomicelle were 49.83 ± 7.65% and 57.79 ± 8.94%, respectively with a nanoparticle yield of 86.91 ± 3.63%.

Drug release assay was performed at two different buffer solutions (pH 7.4 and pH 4.5) individually. The cumulative propofol release from COO-PEG-PLDA nanomicelles was shown to reach 100% at pH 4.5 after 30 min, whereas at pH 7.4 the drug release was about 97% after 120 min (Fig. 2). Obviously, the nanomicelles showed a higher drug release ratio in the acidic microenvironment while a lower propofol release in the physiological pH. Indeed, the propofol-COO-PEG-PLDA nanomicelles are stable in physiological condition, limiting the release of propofol. However, the nanomicelles can dissolve into the acidic solution, which destabilizes different bonds cleavage, therefore the nanomicelles are destabilized and increase the release rate of propofol.

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Fig. 2

The in vitro cumulative propofol released from the COO-PEG-PLDA nanomicelles in different pH media over a time period of 120 min. Data are reported as mean ± SD (n = 3).

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3.3 MTT assay

The in vitro cytotoxic activity of propofol and propofol- COO-PEG-PLDA nanomicelles against PBMCs was measured by the MTT assay at serial concentrations. It is displayed in Fig. 3 that the cytotoxic activity of the both propofol and propofol- COO-PEG-PLDA nanomicelles was dose dependent. In fact, as long as the concentration enhanced, the percentage of cell viability reduced. However, the cytotoxic effect was different for propofol and propofol- COO-PEG-PLDA nanomicelles; the percentage of viable PBMCs incubated with free propofol has reduced to 50% at a concentration of 200 μg/mL. However, the propofol- COO-PEG-PLDA nanomicelles was less cytotoxic, with the concentration causing 50% of viable population equal to >200 μg/mL.

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Fig. 3

Cytotoxic effects of propofol and propofol-COO-PEG-PLDA nanomicelles at different propofol concentrations ranging from 1 to 200 μg/mL, which were incubated with PBMCs at 37 °C for 24 h (n = 3). *P < 0.05, ** P < 0.01, ***P < 0.001.

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3.4 Hemolytic assay

The free propofol revealed a dose-dependent hemolytic activity which enhanced rapidly with the increase of propofol concentration. The hemolysis percentage of propofol reached 44.1% at the propofol concentration of 200 μg/mL, whereas the hemolysis percentage of propofol-COO-PEG-PLDA nanomicelles was around 21% at similar concentration of propofol (Fig. 4). Thus, it can be deduced that COO-PEG-PLDA nanomicelles had superiority in hemocompatibility and could be used as a potential candidate for intravenous administration.

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Fig. 4

Hemolysis percentages of propofol and propofol-COO-PEG-PLDA nanomicelles at different propofol concentrations ranging from 1 to 200 μg/mL, which were incubated with human RBCs at 37 °C for 2 h (n = 3). ** P < 0.01, ***P < 0.001.

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3.5 In vitro permeation assay across rat esophageal epithelium

In order to improve the buccal permeation of topical formulations, in the present study, we demonstrated the efficacy of COO-PEG-PLDA nanomicelles to increase propofol permeation across the rat esophagus epithelium. It was calculated that the COO-PEG-PLDA nanomicelles increased the J_ss by 6.64 times (***P < 0.001) and the Q_5h by 6.01 times (***P < 0.001) (Table 1). Besides the improved permeation, a lack of lag time was also observed, characterizing an immediate transport of the drug.

3.6 Pharmacokinetic assay in vivo

In order to assess the integrity of the COO-PEG-PLDA nanomicelles in vivo, the concentration of propofol in plasma was estimated over the course of 120 min (Fig. 5). The data indicated that the COO-PEG-PLDA nanomicelles provided 2.01-, 1.91-, 2.20-, 2.43- and 1.74-fold increase in plasma concentration of propofol compared to free propofol at different time intervals of 2, 5, 10, 20, and 30 min, respectively. The data also indicated that after 50 min, the concentration of free propofol was zero, whereas COO-PEG-PLDA nanomicelles provided 3.5 mg/mL propofol even after 120 min. These data showed that the pharmacokinetic properties of propofol upon loading into COO-PEG-PLDA nanomicelles greatly increased in comparison with free propofol.

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Fig. 5

Pharmacokinetics of free propofol or propofol- COO-PEG-PLDA nanomicelles in rats. Data are reported as mean ± SD (n = 3).

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3.7 Anesthetic efficacy

In the present study, induction time of anesthesia (Fig. 6A), anesthesia period (Fig. 6b) and walking time (Fig. 6C) was determined. As shown in Fig. 6, after the injection, induction time of anesthesia (Fig. 6A), anesthesia period (Fig. 6b) and walking time (Fig. 6C) were significantly (**P < 0.01) different from those of free drug, indicating the improvement of the anesthetic efficacy of propofol in the form of nanomicelle. Indeed, the encapsulation of propofol in COO-PEG-PLDA nanomicelles increased anesthetic success (**P < 0.01) of propofol.

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Fig. 6

(A) Induction time of anesthesia, (B) anesthesia period, (C) walking time after infiltration of free propofol or propofol- COO-PEG-PLDA nanomicelles in rat paws. Data are reported as mean ± SD (n = 3), ** P < 0.01.

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Based on our result, it was indicated that the COO-PEG-PLDA nanomicelles improved the pharmacokinetic properties of propofol, which may result in a minimization in consecutive injections. It was also found that drug-loaded nanomicelles had lower cytotoxic and hemolytic effects and greater permeation features. There are two probable explanations for this profile. Propofol could be entirely covered by nanomicelles, which can result in the greater permeation of propofol and inhibition the interaction of RBCs with propofol (Shao et al., 2010). In addition, COO-PEG-PLDA nanomicelles could provide potential safety and hemocompatibility due to long-term sustained release and preventing the accumulation of the high concentration of propofol at target site (Liu et al., 2016).

In this study, rat esophageal mucosa was used as a potential target due to close permeability characteristics to those of human tissue. Indeed, esophageal mucosa can be separated very easily and provide a high surface area surface for permeation assays (Del Consuelo et al., 2005). However, it should be noted as the target site of propofol is CNS (Rehberg and Duch, 1999) and the presence of some histological differences between this barrier and blood-brain barrier (Del Consuelo et al., 2005), some experiments should be conducted in the future studies to compare the transport of propofol in the form of free or nano-formulated states across esophageal mucosa and blood brain barrier.

Although we did not assess the different effects of free or nano-formulated states of propofol after topical or intravenous injection, it should be noted that both application approaches can mitigate compound action potentials (CAPs) peak intensity based on nerve conduction. However, the half-maximal inhibitory concentration (IC₅₀) as a marker of propofol’s efficacy depends on the chemical structure and how to use this medicine topically or by injection (Magori et al., 2019).

Different formulations have been developed to date including lipid-based emulsions, non-lipid formulations (such as surfactants, nanoparticle carriers and cyclodextrins), and prodrugs (Baker et al., 2005).

Formulations containing long-chain triglycerides (LCTs) are the most widely available propofol formulations, the most common of which is Diprivan (Sarkar et al., 2016). This formaldehyde has been shown to reduce the common side effects of free propofol and also increases the anesthetic efficacy (Kirkpatrick et al., 1988; Picard and Tramèr, 2000). There is another type of formulation whose lipid content is a combination of medium chain triglycerides (MCTs) and LCT oils. Propoven and propofol-®Lipuro are the most famous emulsions in this category (Kirkpatrick et al., 1988; Kam et al., 2004).

Propofol iodine-free triglyceride microemulsions (Aquafol) contain non-ionic and co-surfactants for emulsifying propofol in water (Lee, 2010). Pharmacokinetic and pharmacodynamic properties of this product are significantly similar to the common formulation (Diprivan), but have more pain during injection (Picard and Tramèr, 2000; Jung et al., 2010). Another lipid-free product is provided which contains sulfobutyl-ether-β-cyclodextrin and water, but this formulation has not been successful in reducing pain during injection (Babu and Godiwala, 2005). By adding the phosphate group to the propofol molecule, propofol can be dissolved in water. So far, two types of propofol phosphorylated prodrugs have been synthesized: propofol phosphate and phosphonooxy methyl propofol 4 (2 and 6 diisopropyl phen-oxymethyl phosphate) (Bengalorkar et al., 2011; Dinis-Oliveira, 2018). The prodrugs must first be converted to propofol in the presence of alkaline phosphatase to begin its sedative effects, which is present in the vascular and hepatic endothelium and is responsible for the conversion of pro-propofol to propofol. Therefore, activation of prodrug takes 1–4 min after injection, while the anesthetic effect of the usual formulation is much faster (Bengalorkar et al., 2011; Dinis-Oliveira, 2018). In general, as these experiments were done in vivo, some pre-clinical and clinical trials are required to further discuss about the potential biomedical application of this nano-based product.