Preparation and Characterization of Sorafenib Nano Emulsion: Impact On Pharmacokinetics and Toxicity An in Vitro and in Vivo Study

Drug Delivery and Translational Research
https://doi.org/10.1007/s13346-024-01530-z
ORIGINAL ARTICLE
Preparation and characterization of Sorafenib nano‑emulsion: impact

on pharmacokinetics and toxicity; an in vitro and in vivo study
Dalia Zaafar1 · Heba M. A. Khalil2 · Gehad E. Elkhouly3,4 · Abanoub Selim Sedeky5,6 · Yasmine H. Ahmed7 ·
Mona G. Khalil1 · Yasmin Abo‑zeid3,4
Accepted: 20 January 2024

© The Author(s) 2024
Abstract
Hepatocellular carcinoma (HCC) ranks as the third leading cause of cancer-related deaths worldwide. Current treatment
strategies include surgical resection, liver transplantation, liver-directed therapy, and systemic therapy. Sorafenib (Sor) is
the first systemic drug authorized by the US Food and Drug Administration (FDA) for HCC treatment. Nevertheless, the
conventional oral administration of Sor presents several limitations: poor solubility, low bioavailability, drug resistance devel-
opment, and off-target tissue accumulation, leading to numerous adverse effects. Nano-emulsion, a nano-delivery system, is
a viable carrier for poorly water-soluble drugs. It aims to enhance drug bioavailability, target organ accumulation, and reduce
off-target tissue exposure, thus improving therapeutic outcomes while minimizing side effects. This study formulated Sor
nano-emulsion (Sor NanoEm) using the homogenization technique. The resultant nano-emulsion was characterized by particle
size (121.75 ± 12 nm), polydispersity index (PDI; 0.310), zeta potential (-12.33 ± 1.34 mV), viscosity (34,776 ± 3276 CPs),
and pH (4.38 ± 0.3). Transmission Electron Microscopy exhibited spherical nano-droplets with no aggregation signs indicat-
ing stability. Furthermore, the encapsulation of Sor within the nano-emulsion sustained its release, potentially reducing the
frequency of therapeutic doses. Cytotoxicity assessments on the HepG2 cell line revealed that Sor NanoEm had a significantly
(P < 0.05) more potent cytotoxic effect compared to Sor suspension. Subsequent tests highlighted superior pharmacokinetic
parameters and reduced dosage requirements of Sor NanoEm in mice. It exhibited an enhanced safety profile, particularly
in behavior, brain, and liver, compared to its suspended form. These findings underscore the enhanced pharmacological and
toxicological attributes of Sor Nano-emulsion, suggesting its potential utility in HCC treatment.
Keywords Hepatocellular Carcinoma · Nano-emulsion · Sorafenib · hepG2 Cell line · Pharmacokinetics
Abbreviations
* Dalia Zaafar ALT Alanine Transaminase
dalia.zaffar@pharm.mti.edu.eg
AST Aspartate Aminotransferase
1
Department of Pharmacology and Toxicology, Faculty Bax Bcl2 associated X protein
of Pharmacy, Modern University for Technology Bcl2 B-cell leukemia/lymphoma 2
and Information, Cairo, Egypt CD31 Cluster of differentiation 31
2
Department of Veterinary Hygiene and Management, Faculty COX-2 Cyclooxygenase 2 (COX-2)
of Veterinary Medicine, Cairo University, Giza 12211, Egypt CYP3A4 Cytochrome P450 3A4
3
Department of Pharmaceutics and Industrial Pharmacy, GGT Gamma-Glutamyl Transferase
Faculty of Pharmacy, Helwan University, Cairo 11795, Egypt HCC Hepatocellular Carcinoma
4
Helwan Nanotechnology Center, Helwan University, LD50 Lethal dose 50
Cairo 11792, Egypt SAP Spontaneous Alternation Percentage
5
Department of Microsystems Engineering (IMTEK), Sor Sorafenib
University of Freiburg, Freiburg im Breisgau, Germany NanoEm Nano-Emulsion
6
Nanomedicine Lab, Center of Materials Science Susp Suspension
(CMS), Zewail City of Science and Technology,
6Th of October 12578, Giza, Egypt
7
Department of Cytology and Histology, Veterinary Medicine
Faculty, Cairo University, Giza 12211, Egypt
Vol.:(0123456789)
Introduction These systems were also reported to overcome drug

resistance, enhance pharmacokinetic parameters, improve
Hepatocellular carcinoma (HCC) stands as the third leading the bioavailability of poorly soluble drugs, regulate drug
cause of cancer-related mortality globally [1]. Despite advance- release, and enhance drug stability [19–23]. Leveraging
ments in cancer surveillance and diagnosis, many patients still nano-delivery systems to transport Sor into the liver actively
have late-stage diagnoses [2–4]. Various treatment strategies for holds promise for improving its therapeutic efficacy while
HCC, including surgical resection, liver transplantation, liver- diminishing its potential side effects.
directed therapy, and systemic therapy, have been employed Several attempts have been made to develop diverse nano-
[5]. Sorafenib (Sor) emerged as the pioneering systemic drug formulations of Sor, including lipid polymer hybrid nanopar-
sanctioned by the US Food and Drug Administration (FDA) for ticles [24], liposomes [25], self-emulsifying drug delivery
HCC treatment [6, 7]. Functioning as a multi-kinase inhibitor, systems [26], cyclodextrin-modified silicon nanoparticles
Sor inhibits serine-threonine kinases Raf-1 and B-Raf, along [27], nanogels [28], diatomite nanoparticles [29], nano col-
with the receptor tyrosine kinase activity of vascular endothe- loidal carrier [30], and pullulan nanoparticles [31]. However,
lial growth factor VEGFRs and platelet-derived growth factor most of these studies focused on non-oral applications [32].
receptor (PDGFR), pivotal elements in the molecular pathogen- The present study aimed to encapsulate Sor into a nano-
esis of HCC [6]. Additionally, atezolizumab plus bevacizumab emulsion for oral delivery. Nano-emulsions, typically sized
garnered FDA approval as a first-line treatment for HCC, exhib- between 20 and 200 nm and composed of oil and aqueous
iting superior survival outcomes compared to Sor [8]. However, phases [28, 33], made it an ideal system for dissolving large
the higher treatment costs associated with these drugs, in con- amounts of lipophilic drugs. Additionally, they enhance
trast to Sor therapy, positioned Sor as a key therapeutic agent drug stability by reducing enzymatic degradation and clear-
for HCC treatment [9, 10]. ance [34]. This research focused on encapsulating Sor into
Despite its efficacy, Sorafenib (Sor) faces several limi- a nano-emulsion to demonstrate that a nano-delivery sys-
tations compromising its effectiveness in cancer therapy, tem applied orally might potentially enhance the therapeutic
including poor solubility, low bioavailability, drug resist- efficacy of Sor and diminish its side effects compared to
ance development, and non-selective action targeting conventional therapy.
healthy and tumor tissues [10–12]. The latter contributes To ascertain our hypothesis, the anticancer efficacy of
to observed side effects during Sor's clinical application, Sor nano-emulsion (Sor NanoEm) was assessed in vitro on
potentially impacting the patient's quality of life [8]. These HepG2 cells and compared to Sorafenib suspension (Sor
side effects may include elevated blood pressure, proteinu- susp), representing the conventional therapy. Subsequently,
ria, hand-foot skin reactions, asthenia, anorexia, diarrhea, the study aimed to determine the lethal dose 50 (LD 50)
and weight loss. Moreover, Sorafenib treatment is often for Sor NanoEm and Sor susp, both in vitro and in vivo
associated with grade 3–4 drug-related adverse events in using male Swiss mice. After investigating the safety of Sor
approximately 50% of patients, leading to withdrawal rates NanoEm compared to conventional therapy, the pharma-
of around 15% [3, 13]. Thus, identifying a delivery sys- cokinetic parameters for Sor susp (100 mg/kg) versus Sor
tem capable of enhancing drug solubility bioavailability, NanoEm (30 mg/kg) were tracked after a single oral dose.
overcoming resistance, and selectively targeting tumor tis- Furthermore, an in vivo study examined animal behav-
sues becomes crucial. Such an improvement could bolster ior for 21 consecutive days following a single oral dose to
its pharmacological activity while reducing side effects, assess the potential side effects of each formulation. This
ultimately enhancing the therapeutic management of HCC. was complemented by histopathological examination and
Nanotechnology, an innovative field utilized for dec- biochemical analysis.
ades, has significantly advanced the efficacy of anticancer
treatments [14]. Nano-delivery systems have been applied
for selective delivery of drugs to diseased organs, mini- Materials and methods
mizing off-target accumulation, this is mainly attributed
to the enhanced permeability and retention effect (EPR), Materials
a pathophysiological condition characterized the tumor,
where tumor vasculature is leaky allowing the accumula- Drugs and chemicals
tion of nano-delivery systems sized less than 200 nm spe-
cifically at the tumor site and overcome their accumulation Sorafenib, generously provided by Professor Tamer Nasr
at organs with normal vasculature (non-leaky) [15, 16]. (Nexavar®, Bayer, USA), was utilized in this study. The
This could be associated with reducing drug frequency pure Sorafenib powder was obtained by grinding the tablets
[17], and subsequently reducing drug side effects [16–18]. and dissolving them in acetonitrile/water (90/10, v/v), as
previously described [35]. Following the filtration to remove pH 1.2 and pH 7.4 was determined over concentrations ranging
drug excipients and additives, the resulting solution under- from 0.04 to 2.5 µg/ml and 0.3 to 10 µg/ml, respectively, where
went evaporation using a rotary evaporator to yield the pure Sor was dissolved in DMSO: PBS (10 mM) at a molar ratio of
Sorafenib powder employed in the present studies. 1:2, followed by sample analysis at λmax 264 nm.
Tris, glycine, SDS, Tween 80, Polyethylene glycol 600 The accuracy and precision of inter-day and intra-day
(PEG 600), Dimethyl sulfoxide (DMSO), phosphate buffer variations were evaluated using Sor concentrations of 0.31,
saline (PBS) tablets, and Castor oil were procured from 1.5, and 2.5 µg/mL at pH 1.2, and 2, 2.5, and 10 µg/ml at
Sigma Aldrich. All chemicals were of analytical grade. The pH 7.4. Each concentration was analyzed three times on the
HepG2 liver cancer cell line was purchased from the Ameri- same day at different intervals (intra-day variation) or on
can Type Culture Collection (HB-8065). separate days (inter-day variation). Accuracy and precision
were computed using Eqs. (1) and (2), respectively.
Methodology
Accuracy = (M − N)∕(N) ∗ 100 (1)
Preparation and characterization of Sor NanoEm M is the mean value of Sor concentration measurements,
while N is the theoretical concentration.
Sor NanoEm was formulated using castor oil, phosphate-
buffered saline (PBS, 10 mM, pH 7.4), Tween 80, and PEG Precision(%RSD) = SD∕M ∗ 100 (2)
600 at 10:5:2.5:2.5, respectively. Initially, Sorafenib pow-
RSD is the relative standard deviation, SD is the standard
der (40 mg) was dissolved in PEG 600 (as a co-surfactant)
deviation of measurements, and M is the mean value of Sor
and Tween 80 (as a surfactant). Subsequently, castor oil was
measurements.
added, and the mixture was ultrasonicated in a water bath
sonicator (Elmasonic S 30 H, Germany) for 5 min. Fol-
Release study
lowing this, the blend was placed in a cold-water bath, and
a solution of PBS (10 mM, pH 7.4) was gradually added
Sor NanoEm and Sor solution (Sor pure powder dissolved
dropwise under homogenization (Daihan Homogenizer, ZS
in DMSO) were investigated for drug release using a modi-
version, Korea) at 750 rpm for 11 min. The formed nano-
fied version of the protocol outlined in previous studies
emulsion contained Sor at a concentration 2 mg/ml was used
[30, 32, 36]. In this study, NanoEm or Sor DMSO solution
for characterization and other experimental work.
(1 ml, 2 mg/ml) was placed within a dialysis membrane bag
The Sor NanoEm droplet size, PDI, and zeta potential
(Dimension; 5 cm × 2.5 cm, pore size 2.4 nm, molecular
were assessed using a Malvern Zeta-sizer Nano ZS (Malvern
weight cutoff 12–14 kDa) that had been pre-conditioned
Instruments Ltd, Malvern, UK) at a constant temperature of
with the release medium [DMSO: PBS (10 mM, pH 1.2)
25 °C ± 0.1. Samples were diluted in PBS (10 mM, pH 7.4)
(1:2)] for 24 h. The release experiment involved an initial
to maintain a count rate ranging from 50 to 300 KCPs. For
2-h duration simulating gastric pH, followed by reloca-
morphological analysis, Transmission Electron Microscopy
tion of the dialysis membrane to another release medium
(TEM) (H-700, Hitachi Ltd., Japan) was employed where
[DMSO: PBS (10 mM, pH 7.4) (1:2)] to simulate intestinal
samples were diluted (1:50) with PBS (10 mM, pH 7.4). A
pH. Drug release was monitored for up to 48 h under con-
drop of the diluted solution was applied to a mesh copper
tinuous stirring (100 rpm) at 37°C. Samples (3 mL) were
grid coated with carbon film. After drying for 5 min, a drop
periodically withdrawn and replaced with an equal volume
of phospho-tungstic acid (2% w/w) was added to the grid for
of fresh-release medium at specific intervals. Each time-
50 s, followed by removal of excess liquid using filter paper.
point was run in triplicate and analyzed by UV–Visible
Additionally, the pH and viscosity of the nano-emulsion
spectrophotometry at λmax 264 nm.
were measured at room temperature. pH was determined
using an Ohaus Economical pH bench meter (Starter 3100,
USA), calibrated with three standard buffer solutions (pH Release kinetics
4, 7, and 10). Viscosity was assessed using Lamy Rheology
(B-one Plus, Germany) with spindle 6 at 50 rpm for 3 min. The kinetics of Sor release from the nano-emulsion was
assessed using DD Solver software, following the method-
UV–Visible spectrophotometric analysis and its validation ology reported in earlier studies [34, 36]. The release data
underwent fitting into various release kinetic models, and
The UV–Visible Spectrophotometric analysis of Sor was the model that exhibited the highest coefficient value of R2
validated, following our established protocol (Abo-zeid et al. was chosen as the most suitable mathematical representation
2022), with the following modifications. The linearity of Sor at for the kinetic release profile.
Cell growth inhibition and cytotoxic effect of Sor‑Susp Lethal dose 50 determination
and Sor‑NanoEm using MTT assay on HepG2 cell line
In this study, we initially determined the LD50 of the
Cell line culture Cells were cultured in DMEM (Invitrogen/ Sor-NanoEm formulation. Mice underwent a 12-h fast-
Life Technologies) supplemented with 1% penicillin–strep- ing period before receiving a single oral administration
tomycin and maintained in a humidified atmosphere with of Sor-NanoEm.
5% CO2 at 37°C. The experiments were replicated in three
independent trials. Estimation of the dose range and percentage of mortali‑
ties The experiment started with a gavage administration of
MTT cytotoxicity assay The MTT assay involved the deter- a single oral dose of Sor-NanoEm (150 mg/kg) to mice the
mination of the percentage of cell viability after incuba- following day after a prior evening fast. Mice were individu-
tion with tested samples, followed by calculating the IC50 ally placed with access to food and water for the subsequent
value for each sample to compare their efficacy in inhibiting two hours. To establish the LD50, a modified 4-level Up and
HepG2 cells, following established protocols [35, 37]. Down Procedure was employed, as previously reported [39].
The effect of Sor Susp and Sor NanoEm on cell prolif- This involved an increase in the number of mice at each
eration was evaluated using the MTT (Cat. no. 298–93- subsequent level: three mice at the first level, five at the
1; Cyman Chemical Co., USA) uptake method. Around second, seven at the third, and so forth. If over 50% of the
3 × 103 cells were seeded per well in a 96-well plate and mice died, the subsequent level dose was reduced. Con-
incubated for 12 h. The following day, cells were exposed versely, if fewer than 50% of the mice succumbed, the dose
to varying Sor Susp and Sor NanoEm concentrations at was increased. Signs were meticulously recorded at each
37°C for 48 h. Subsequently, MTT reagent (5 mg/ml; dosage level throughout the experiment.
Sigma Aldrich) was added to each well and incubated at
37°C for 4 h. The absorbance was measured at 450 nm Pharmacokinetics study
using a ROBONIK P2000 Elisa Reader (Thermo Fisher
Scientific, Inc.). The mice were divided into two groups, each consist-
ing of 15 animals. Group I received Sor-NanoEm at a
In‑vivo mice experimental studies concentration of 30 mg/kg. In comparison, Group II was
administered free Sor powder suspended in 2.5% carbox-
Animal housing and Statement of ethics ymethyl cellulose (CMC) at a dose of 100 mg/kg via oral
gavage. Blood samples (200 µL) were collected from the
The experimental procedures adhered to the NIH Guide- retro-orbital plexus of the mice using heparin-containing
lines for the Care and Use of Laboratory Animals and tubes at specified time intervals. Plasma proteins were
were approved by the Institutional Animal Care and Use precipitated from the blood samples by centrifugation at
Committee at the Faculty of Veterinary Medicine, Cairo 12,000 rpm for 10 min. Subsequently, Sor levels in the
University (Approval number: Vet CU 09092023768). extracted plasma samples were estimated using the fol-
All procedures were conducted following the ARRIVE lowing HPLC method.
guidelines to ensure the utmost care for the animals used
in the research [38]. High‑performance liquid chromatography (HPLC) assay
Male Swiss mice weighing 25–27 g were procured from
a commercial supplier (National Cancer Institute, Cairo, The concentrations of Sor were measured using high-perfor-
Egypt) for all in-vivo experiments, including determin- mance liquid chromatography (HPLC) techniques equipped
ing lethal dose 50, performing pharmacokinetic study, and with ultraviolet (UV) detection (HPLC system Echromatecg
determining the main biological parameter level. Mice were Mod. C 1620 liquid chromatography).
acclimatized for one week before the commencement of the A reversed-phase C18 column facilitated the gradient
experiments and housed in plastic shoebox-type cages (5 elution of the mobile phase from water (eluent A) to ace-
mice/ cage, 27 × 13 × 13 cm) provided with coarse saw dust tonitrile (eluent B) at a flow rate of 1.0 mL/min. The gradi-
as a bedding material. They were maintained under standard ent transitioned linearly from 60% eluent A and 40% eluent
laboratory conditions (temperature: 25˚C; humidity: 50%; B to 17.5% eluent A and 82.5% eluent B (Brocks et al.,
normal light/dark cycle) and had ad libitum access to a well- 2010). Subsequently, all measured parameters underwent
balanced commercial diet and water throughout the study. statistical analysis.
Evaluation of Pharmacokinetic Parameters Behavioral study
The R® software version 3.5.2 was employed to calculate After completing the final dose of treatment, all mice
various pharmacokinetic parameters, including the area were placed in the behavioral analysis room and allowed
under the plasma concentration–time curve from zero to to acclimate to the presence of the experimenter for 2 h.
the time of the last measurable concentration (AUC0-t), Subsequently, the mice underwent assessments to evaluate
the area under the plasma concentration–time curve from sickness-like behaviors, encompassing anxiety, depression,
zero to infinity (AUC0-∞), maximum plasma concentra- and cognitive deficits through distinct behavioral tests:
tion (Cmax), elimination rate constant (Kel), half-life in
the elimination phase ( t1/2), apparent plasma drug clear- Anxiety‑like behavior using open field test and dark–light
ance (Cl), and apparent volume of distribution (Vd). activity box
In‑vivo investigation of the impact of Sor‑NanoEm Mice underwent an open-field test, a standard method for
on mice's behavioral and biochemical parameters assessing rodent locomotion and exploratory behavior
[41]. Each mouse was placed in one corner of a wooden
Study design Twenty-four male albino mice were randomly box divided into 16 squares, and its activity was recorded
distributed into four groups (n = 6) as follows: for 3 min. Measured parameters included the number of
squares crossed and rearing frequency [42]. Additionally,
Group I: Control—Mice received orally administered a dark–light activity box test was conducted, where mice
2.5% CMC solution (2 ml/kg/day), the vehicle used to spent 5 min in a box with two chambers: one dark and one
suspend Sor to formulate Sor Suspension. illuminated. The parameters assessed were the frequency
Group II: Blank—Mice were orally administered Blank and duration of entries into light or dark chambers [43].
nano-emulsion (2 ml/kg/day), (nano-emulsion prepared
as described in section 'Preparation and characterization Depressive‑like behavior using forced swim test
of Sor NanoEm' but in the absence of Sor).
Group III: Sor-Susp group—Mice were orally administered The forced swim test is a screening tool for assessing the
Sor suspended in 2.5% CMC (100 mg/kg daily) [13, 40]. antidepressant activity of drugs [44]. Mice were individually
Group IV: Sor-NanoEm group—Mice were orally admin- tested for 6 min in a warm, water-filled cylindrical container.
istered a daily dose of Sor-NanoEm formulation (30 mg/ The initial 2 min of the video recording were excluded, and
kg daily). the subsequent 4 min were assessed for mobility and immo-
bility duration. Mobility behaviors such as swimming, div-
The treatment for each group continued for 21 consecutive ing, and climbing against the bottle wall were considered,
days, as illustrated in Fig. 1. while immobility referred to minimal movements to keep
Fig. 1 A flow chart showing

the experimental design of the
in vivo study
the mouse's head above the water. After the test, each mouse Elabscience (USA) for GGT (Catalog number #E-EL-
was dried in a wooden chip-filled cage before returning to R0404), and LifeSpan BioSciences, Inc. (Washington, USA)
its original cage. Fecal pellets were removed between each for CYP 3A4 (Catalog number #LS-F27379). The concen-
mouse, and water changes were made if necessary [45]. tration of each enzyme was evaluated by measuring the opti-
cal density using an automated ELISA reader set at 450 nm.
Cognitive deficits using the Y‑maze test
Estimation of hepatic tissue levels of Bcl2 and Bax using the
The Y-maze test assesses spatial working memory, reli- ELISA technique Sandwich enzyme-linked immunosorbent
ant on hippocampal integrity. Each mouse was placed in assay (ELISA) kits from Cloud-Clone Corp were employed
a Y-shaped wooden maze and tested for 5 min. Parameters to assess the levels of B-cell leukemia/lymphoma 2 (Bcl2)
measured included the number of arm entries and the spon- and Bcl2 associated X protein (Bax) in liver tissue homoge-
taneous alternation percentage (SAP). SAP was computed nate, following the manufacturer's instructions (Catalog
using the formula [40, 42]: number #SEA778Ra for Bcl2 and #SEB343Ra for Bax),
(Cloud-Clone Corp. (Wuhan, China).
SAP = numberofalternations∕(totalarmentries − 2) × 100
Histopathological examination The liver, cerebrum, and
hippocampus specimens were collected and fixed in 10%
Euthanasia, blood, and tissue sampling neutral buffered formalin for 48 h. Subsequently, they were
dehydrated using increasing concentrations of ethyl alcohol,
After the conclusion of the behavioral tests, mice were cleared with xylene, and embedded in paraffin wax. Sections
euthanized using an intraperitoneal injection of a ketamine of 4 µm thickness were prepared, deparaffinized, and stained
(100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) mixture [46]. with hematoxylin and eosin (H&E) for examination under a
Blood samples were collected from the inner canthus of each light microscope [47].
mouse's eye and centrifuged at 3000 rpm for 15 min to obtain
separated sera, which were stored at -80°C until liver func- Immunohistochemical examination
tion parameters were evaluated. Subsequently, the mice were
euthanized by cervical dislocation. The liver and brain were Cyclooxygenase 2 protein (COX‑2) immunostain‑
harvested and rinsed with cold saline. Half of each organ ing Cyclooxygenase 2 (COX-2) (Catalog # PA1-37,505,
was preserved at -80°C for biochemical analysis, while the Thermos Fisher Scientific, USA) was employed in the pre-
remaining halves were fixed in 10% formalin-buffered saline sent study to identify liver, cerebral cortex, and hippocampus
for histopathological and immunohistochemical examinations. inflammation. The method used was outlined according to
Tissue homogenates of the brain and liver were prepared Côté and colleagues [48].
in phosphate buffer (0.05 M, pH 7) using a polytron homog-
enizer at 4°C. The homogenate underwent centrifugation at Cluster of differentiation 31 (CD31) immunostaining Cluster
10,000 rpm for 20 min to eliminate cell debris, unbroken of differentiation 31 (CD31) (Catalog # 14–0311-82, Thermos
cells, nuclei, erythrocytes, and mitochondria. The resulting Fisher Scientific, USA) was used in the current study to iden-
supernatant (cytoplasmic extract) was utilized for subsequent tify vascular lesions in the liver and cerebrum. The method
biochemical analysis following the manufacturer's guidelines. used was outlined according to Hsu and colleagues [49].
Fixed tissue samples were dehydrated, treated with
xylene, and embedded in paraffin. Sections of 3μm thickness Image analysis The assessment of immunohistochemical
were generated, deparaffinized, and stained with hematoxy- observations (area percentage) involved analyzing sections
lin and eosin (H&E) for histological examination. stained with anti-COX-2 and anti-CD31 using a digital Leica
Quin 500 image analysis system (Leica Microsystems, Swit-
Biochemical analysis zerland) located at the Faculty of Dentistry, Cairo Univer-
sity. Statistical analysis was conducted on each specimen's
Evaluation of different liver enzymes levels Serum levels mean values and standard deviations.
of various liver enzymes—alanine aminotransferase (ALT),
aspartate aminotransferase (AST), Gamma Glutamyl Trans- Statistical analysis
ferase 1 (GGT)—as well as levels of Cytochrome P450 3A4
(CYP3A4) in liver tissue were determined using the ELISA The data analysis was performed using GraphPad Prism 9.0
sandwich technique following the protocols provided by software. Mean values and standard deviations were utilized
BIOMATIK (Ontario, Canada) for ALT (Catalog number to represent the experimental findings. IC50 values were
#EKU02211) and AST (Catalog number #EKE62019), calculated using sigmoid nonlinear regression [50]. For data
Table 1 Physicochemical Sample name Droplet Size Zeta Potential PDI pH Viscosity (Cps)
characterization of Sor NanoEm (D nm ± SD) (mv ± SD)
and blank Nano-Em
Sor-NanoEm 121.75 ± 12 - 12.33 ± 1.34 0.310 4.38 ± 0.3 34,776 ± 3276
Blank 80.33 ± 29.78 - 11.37 ± 2.84 0.330 4.68 ± 0.2 30,111 ± 4197
analysis, one-way variance analysis (ANOVA) and the post as the average value of droplet size diameter (nm) ± stand-
hoc Tukey test for multiple comparisons were employed. Sta- ard deviation (SD), and it was 121.75 ± 12 nm that was sig-
tistical significance was set at a P-value below 0.05. nificantly (P < 0.05) larger than the size recorded for blank
Nano-Em, 80.33 ± 29.78 nm. The PDI values for the formed
Results Sor Nano-Em indicated a monodisperse sample where the
PDI value was ≤ 0.3, and it was close to the PDI value of
Preparation and characterization of Sor‑NanoEm Blank Nano-Em (0.330).
The values of zeta potential recorded for Nano-Em
The formed Sor-NanoEm were prepared and characterized were presented as zeta potential (mv) ± SD, and it was—
as described in section ‘Drugs and chemicals’, and the data 12.33 ± 1.34mv for Sor NanoEm, was close to the zeta poten-
obtained was presented in Table 1. Droplet size was shown tial value of blank Nano-Em,—11.37 ± 2.84 mv. The low
Fig. 2 TEM image of Sor-

NanoEm, scale bar 100 nm
Fig. 3 In vitro release profile of

Sor suspension (Sor-Susp) and Sor Nano_Em
Sor-NanoEm over 48 h where
release medium was DMSO: Sor Solution
PBS (10mM, pH 1.2) for the
100
% Cumulative Drug Release

first 2 h, then after, release study
was continued in DMSO: PBS
(10mM, pH7.4) at 37°C ± 0.5°C
80
60
40
20
0
0 10 20 30 40 50
Time (hr)
negativity values might be attributed to using a non-ionic sur- from 0.36 to 15.39 and 0.88 to 52.48, respectively. The preci-
factant, Tween 80, as an emulsifying agent [51]. sion (%RSD) was also calculated, ranging from 0.02% to 0.2%
The viscosity recorded for Sor-NanoEm was 34,776 ± 3276 and 2.5% to 20.12% for intra-day and inter-day variations,
Cps, and it was non-significantly (P > 0.05) higher than the respectively. For analysis at pH 7.4, the accuracy determined
viscosity recorded for blank Nano-Em, 30,111 ± 4197. pH for intra-day and inter-day variations was 1.78 to 11.79 and
recorded for Sor-NanoEm and Blank Nano-Em was 4.38 ± 0.3 from 1.94 to 11.59, respectively. The precision values deter-
and 4.68 ± 0.2, respectively. mined for Sor analysis at pH 7.4 were 0.02 to 0.17 and from
Transmission electron microscopy images of Sor-NanoEm 2.5 to 20.17 for intra-day and inter-day variations, respectively.
were presented in Fig. 2. Images showed spherical Nano-Em
with no sign of aggregation, and the droplet size ranged from Release study
60.9 to 102 nm.
A release study for Sor suspension and Sor-NanoEm was
UV–Visible Spectrophotometric analysis performed as described in section ‘UV–Visible spectrophoto-
and its validation metric analysis and its validation’. The dialysis bag diffusion
technique presented release graphs in Fig. 3. As discussed, the
The validation of UV–visible spectrophotometric analysis of cumulative Sor release percentage was determined by UV–vis-
Sor was performed, as described in section ‘Preparation and ible spectrophotometric analysis. As revealed in Fig. 3, the
characterization of Sor NanoEm’. The linearity of Sor at pH cumulative release percentage after 8h for Sor solution was
1.2 and 7.4 was demonstrated over a concentration range from 100%, consistent with the literature. Contrary to Sor Nano-Em,
2.4 ng to 2.5 µg and 0.3 to 10 µg/mL, and the correlation coef- drug release percentage at 8 and 10h was 53% and 87.93%,
ficient (R2) values were 0.9931 and 0.9978, respectively. The respectively. The release of Sor was plateaued after 10 h for
values of the limit of detection (LOD) and limit of quantifica- Sor-NanoEm, reflecting the sustained release properties from
tion (LOQ) at pH 1.2 were 0.279 and 0.846, while their values Sor Nano-Em.
at pH 7.4 were 1 and 3 μg/mL, respectively. Release kinetic models were applied to assess the mech-
The intra-day and inter-day variations at pH 1.2 were per- anism of Sor release from Sor-NanoEm. R2 values were
formed using three concentrations (0.321, 1.5, and 2.5 μg/mL). obtained by linear regression analysis, and they were the best-
The accuracy for intra-day and inter-day variations ranged fitting zero-order kinetics for Sor-NanoEm.
Cell growth inhibition and Cytotoxic effect The cell viability percentage exhibited dose-dependency,
of Sor‑Susp and Sor‑NanoEm using MTT assay with an increase in Sor concentration correlating with a
on HepG2 Cell line decrease in cell viability percentage. However, a signifi-
cantly (P < 0.05) more significant reduction in cell viability
The viability percentage of HepG2 cells treated with Sor percentage was observed with Sor NanoEm compared to
suspension and its Nano-Em forms is depicted in Fig. 4A. Sor suspension.
Fig. 4 MTT cytotoxicity study of Sor Nano-Em on HepG2 liver can- asterisks * at P < 0.05. B Determination of cytotoxic concentration
cer cell line; A Cell viability percentage of HepG2 after treatment (µg/ml) responsible for the death of 50% of HepG2 cells (IC50) for
with sorafenib suspension (Sor Susp) and Sor NanoEM. Significant Sor Susp and Sor NanoEm formulas
differences in the cytotoxic effect on HepG2 cells are denoted with
Fig. 7 Effect of Sora- Susp and Sor Nano-Em administration on the anxiety- ◂
like behavior of the mice. a Open field test (number of crossing squares), b
Open field test (Rearing frequency), c Light dark activity box (light cham-
ber duration), d Light dark activity box (light chamber entries), e Light dark
activity box (dark chamber duration), and f Light dark activity box (dark
chamber entries). One-way ANOVA followed by Tukey's post hoc test was
used. * Significantly differs from the control group; #significantly differs
from the blank group; x significantly differs from Sor-Susp at p < 0.05
Severe diarrhea accompanied by shivering was promptly

observed after the administration of toxic doses (ranging
from 50 to 150 mg/kg). Additionally, convulsions became
increasingly visible and hazardous over time. When the 30
mg/kg dose was administered, No Observed Adverse Effect
Level (NOAEL) was reached, as all mice survived.
Fig. 5 Dose–response mortality curve of oral Sor Nano-Em in mice.
Figure 5 illustrates these observations on a scale depicting
Percentage lethality values are plotted against the concentration of the
formula. The LD50 value is indicated the percentage of mouse mortality versus the administered
dose. Based on this data, a nonlinear regression fitting pro-
cedure was utilized to estimate the oral LD50 and LD100 to
To determine the enhanced cytotoxic effect of Sor Nano- be 48 mg/kg and 150 mg/kg, respectively.
Em over Sor suspension, IC50 values were calculated for The onset of toxic signs and symptoms following oral
both formulations using the dose–response curve presented administration of Sor NanoEm displayed dose dependency.
in Fig. 4B. The results indicated an IC50 value of 4.73 µg/ml At a dose of 150 mg/kg, poisonous symptoms appeared two
(r2 = 0.85) for Sor NanoEm and 11.14 µg/ml (r2 = 0.88) for Sor hours post-administration, primarily involving diarrhea and
suspension. These findings highlight the superior cytotoxic symptoms related to CNS stimulation. However, at doses of
activity of Sor Nano-Em against HepG2 cells compared to 75 and 50 mg/kg, toxicity signs emerged between 6 to 12 h
Sor suspension. after drug administration, characterized by trembling behav-
iors such as chewing, licking, rolling, arching, shivering, and
Toxic lethal dose 50 determination coarse whole-body tremors. Subsequently, animals displayed
signs of distress, such as gasping and labored breathing, ulti-
All mice administered 150 mg/kg of Sor-NanoEm via oral mately leading to mortality.
gavage died within one hour of the experiment. Subse- Conversely, no observable signs of toxicity were evident
quently, a 75 mg/kg dose was tested, resulting in the death after oral administration of 30 mg/kg of Sor NanoEm. No
of three out of four mice. Following this, a 50 mg/kg dose
was administered, leading to the death of a single mouse.
Finally, 30 mg/kg was given, with all animals surviving. Table 2 Plasma pharmacokinetic parameters of sorafenib suspension
(100 mg/kg) and Sor Nano-Em (30 mg/kg) after the administration of
a single oral dose
Pharmacokinetic Parameter Sor NanoEm Sor Susp
C max (µg/mL) 17.33 ± 0.24 9.6 ± 0.1

AUC0-t (µg.h/mL) 88.9 ± 0.16 58.4 ± 0.21
AUC0-∞(µg.h/mL) 232.2 ± 0.21 23.27 ± 0.145
Kel (h−1) 0.0135 ± 0.0008 0.051 ± 0.0011
T ½ (h) 50.93 ± 0.174 12.96 ± 0.177
Vd (mL) 7.2 ± 0.17 24.02 ± 0.186
CL (L/h) 0.112 ± 0.0095 1.111 ± 0.07
AUC0-t, area under the plasma concentration–time curve from zero

to the time of last measurable concentration; AUC0–∞, area under
the plasma concentration–time curve from zero to infinity; Cmax,
maximum plasma concentration; Kel, elimination rate constant; t1/2,
half-life in the elimination phase; Cl, apparent plasma drug clear-
Fig. 6 The sorafenib plasma concentration–time profiles in mice ance; Vd, apparent volume of distribution. Data were presented as
receiving sorafenib suspension (Sor-Susp) (100 mg/kg) and Sor mean ± SD, bold font represented significant difference when P is less
NanoEm (30 mg/kg). Data were presented as mean ± standard deviation than 0.05
Fig. 8 Effect of Sora- Susp and Sor Nano-Em administration on the percentage). One-way ANOVA followed by Tukey's post hoc test was
mice's depressive-like behavior and spatial working memory. a Forced used. * Significantly differs from the control group; #significantly differs
swim test (Mobility duration), b Forced swim test (Immobility), c from the blank group; x significantly differs from Sor-Susp at p < 0.05
Y-maze (number of arm entries), and d Y-maze (Spontaneous alternation
adverse effects on the CNS were observed at this dosage, of arm entries, accompanied by a significant (P < 0.05) decrease
and all animals remained alive. in the SAP compared to the control mice. Conversely, mice
treated with Sor Nano-Em displayed a considerable increase in
Pharmacokinetics Study arm entries, corresponding to a significant (P < 0.05) rise in the
SAP compared to those treated with Sor Susp.
The plasma concentration–time curve after administering a
single oral dose of Sor susp (100 mg/kg) and Sor NanoEm Effect on levels of liver enzymes
(30 mg/kg) was depicted in Fig. 6. Corresponding pharma-
cokinetic parameters for each Sor formulation were pre- Figure 9 illustrates the liver enzyme levels across the vari-
sented in Table 2. The data obtained revealed a significant ous tested groups. The serum level of ALT in mice treated
(P < 0.05) increase in the drug's Cmax, AUC0-t, AUC0-∞, with Sor Susp displayed a significant (P < 0.05) increase,
and half-life but demonstrated a reduction of Kel, Vd, and recording the highest value among all groups. Conversely,
CL for Sor NanoEm compared to Sor susp. mice treated with Sor Nano-Em showed normal levels of
all evaluated enzymes. Additionally, the measurement of
Behavioral analysis cytochrome P450 3A4 levels in liver tissue revealed non-
significant (P > 0.05) differences among most groups, except
Anxiety like behavior for mice treated with Sor Susp, which displayed a marked
increase in CYP 3A4 levels in liver samples (Fig. 9).
As depicted in Fig. 7, the open field test revealed a significant
(P < 0.05) reduction in motor activity in mice treated with Sor Effect on liver tissue levels of Bax and Bcl2
susp. This was evident from the decrease in crossing squares and
rearing frequency compared to the control group. Conversely, The data indicated that the group treated with Sor Susp
mice treated with Sor Nano-Em displayed a notable increase displayed the highest level of Bax protein. However, this
in general motor activity, characterized by a marked rise in level did not significantly (P > 0.05) differ from the levels
crossing squares and rearing frequency compared to Sor susp- observed in other groups (Fig. 10a). Conversely. In contrast,
treated mice. A non-significant (P > 0.05) difference between the the Bcl2 levels among animals treated with blank, Sor Susp,
control and blank groups indicated that the observed behavioral and Sor NanoEm were not significantly (P > 0.05) different;
changes were primarily due to Sor administration.
In the dark–light activity box test, mice treated with Sor
susp exhibited a marked reduction in the frequency of enter-
ing the light chamber. They spent less time there, accompa-
nied by a significant (P < 0.05) increase in the frequency of
entering the dark chamber and more time spent within it than
the control group. Conversely, Sor Nano-Em administration
was associated with increased frequency of entering and time
spent in the light chamber. Moreover, there was a decrease in
the frequency of entering the dark chamber and reduced time
spent in it compared to mice treated with Sor susp.
Depressive like behavior
As illustrated in Fig. 8a and b, the forced swim test indicated

that mice treated with Sor Susp showed a considerable decrease
in mobility duration and a notable increase in immobility dura-
tion compared to control mice. In contrast, mice treated with
Sor NanoEm exhibited a significant (P < 0.05) increase in
mobility duration coupled with a marked reduction in immo-
Fig. 9 Effects of administering sorafenib suspension in 2.5% CMC
bility duration compared to those treated with Sor Susp. (Sor-Susp) or Sor-NanoEm on serum levels of the enzymes: alanine
transaminase (ALT, ng/ml), aspartate transaminase (AST, ng/ml),
Effect on the Cognitive deficits Gamma-glutamyl transferase (GGT, ng/ml) and liver tissue levels of
cytochrome P3A4 (CYP3A4, ng/mg protein) using one-way ANOVA
followed by Tukey's post hoc test. * Significantly differs from the
The results from the Y-maze test (Fig. 8c and d) indicated that control group; #significantly differs from the blank group; x signifi-
mice treated with Sor Susp exhibited a reduction in the number cantly differs from Sor-Susp at p < 0.05
Fig. 10 Effects of administer-

ing sorafenib suspension in
2.5% CMC (Sor-Susp) or Sor-
NanoEm on liver tissue levels
of the apoptosis biomarkers;
A: Bax (ng/mg protein) and
B: Bcl2 (ng/mg protein) using
one-way ANOVA followed by
Tukey's post hoc test. * Signifi-
cantly differs from the control
group at p < 0.05
they were significantly lower (P < 0.05) than the Bcl2 levels as dilated and congested hepatic sinusoids, a congested cen-
recorded for the control group (Fig. 10b). tral vein, cytoplasmic vacuolar degeneration in hepatocytes,
and pyknotic nuclei in some hepatic cells compared to the
Histopathological examination control (Fig. 11c). However, the livers of mice treated with
Sor Nano-Em showed fewer histo-architectural alterations,
Light microscopic observations with less disorganization of hepatic cords and fewer dilations
of hepatic sinusoids (Fig. 11d).
Histological examination of liver tissues from the con- H&E-stained sections of the cerebral cortex from the
trol and blank groups displayed a regular arrangement of control and blank groups revealed a typical structure and
hepatic cords radiating from a central vein and separated by distribution of neurons, neuroglia, and neuropil (Fig. 12a
hepatic sinusoids (Fig. 11a & b). In contrast, liver sections & b). In contrast, the cerebral cortex of mice treated with
of mice treated with Sor-Susp exhibited abnormalities such Sor-Susp showed neuropil vacuolation, congested blood
capillaries with perivascular space, and pyknotic pyramidal with Sor Nano-Em showed only a few pyknotic pyramidal
neurons with pericellular space (Fig. 12c). However, in the neurons with pericellular space compared to the Sor-Susp
cerebral cortex of mice treated with Sor Nano-Em, only a group (Fig. 12h).
few degenerated pyramidal neurons with pericellular space
and vascular congestion were observed compared to those Immunohistochemical observations
treated with Sor susp (Fig. 12d).
Both the hippocampus sections of the control and blank Immunohistochemical examination of COX-2 in the
groups showed standard architecture of the three layers: liver, cerebral cortex, and hippocampus of the control
molecular, pyramidal, and polymorphic. These layers con- (Fig. 13a, e, & i) and blank (Fig. 13b, f, & j) groups dis-
sisted of neurons, neuroglia, and blood capillaries (Fig. 12e played negative cytoplasmic COX-2 immune-expression.
& f). However, hippocampal sections from mice treated with However, liver tissue, cerebral cortex, and hippocam-
Sor-Susp exhibited congested blood capillaries with perivas- pus of mice treated with Sor-Susp revealed significant
cular spaces in the molecular and polymorphic layers and positive cytoplasmic COX-2 immunoreactivity in some
disarrangement of pyramidal cells in the principal cell layer, hepatocytes and neurons (Fig. 13c, g, & k and Fig. 14).
including some pyknotic neurons with pericellular spaces Conversely, mice treated with Sor Nano-Em displayed a
(Fig. 12g). In contrast, the hippocampus of mice treated significant reduction in COX-2 immuno-expression in
Fig. 11 a:d liver sections of adult male albino mice. H&E X400. a arrow), vacuolar degeneration of hepatocyte cytoplasm (red arrow),
& d liver of control and blank groups showed regular hepatic cords and nuclei pyknosis of some hepatocytes (circle). d The livers of
(H), central vein (CV), and hepatic sinusoids (arrow). c) The liver sorafenib nano-emulsion-treated mice showed disorganization of
of sorafenib suspension-treated mice revealed a congested central hepatic cords (circle) and dilation of hepatic sinusoids (arrow)
vein (yellow arrow), dilated and congested hepatic sinusoids (black
Fig. 12 a: h Brain tissue of adult male mice. H&E X400. a & b cer- pyramidal cell layer (P) that formed of triangular neurons with vesic-
ebral cortex of control and blank mice showed standard structure and ular and spherical neurons (yellow arrow) and the polymorphic layer
distribution of neurons, neuroglia, and neuropil. c The cerebral cortex (PL) that consisted of neurons and neuroglia (green arrow). g hip-
of sorafenib suspension-treated mice revealed congested blood cap- pocampus of sorafenib suspension-treated mice revealed congested
illaries with perivascular spaces (yellow arrow), pyknotic pyramidal blood capillaries with perivascular space (black arrow), disarrange-
neurons (black arrow), and neuropil vacuolation (blue arrow). d The ment of pyramidal cell layer neurons (arched arrow), some pyrami-
cerebral cortex of mice treated with sorafenib nano-emulsion showed dal cells appeared pyknotic with perivascular spaces (yellow arrows).
vascular congestion (yellow arrow) and pyknotic pyramidal cells with h The hippocampus of mice treated with sorafenib nano-emulsion
pericellular space (black arrow). e & f hippocampus of control and showed few pyknotic pyramidal cells with perineural space (yellow
blank mice exhibited standard structure of the molecular layer (M) arrow)
composed of neurons (red arrow) and neuroglia (black arrow), the
the liver and a non-significant reduction in neurons of Discussion

the cerebral cortex and hippocampus compared to the
Sor-Susp group (Fig. 13d, h, & I and Fig. 14). Sor is considered a first-line medicinal agent to manage
In the liver of control and blank mice, there was mild HCC. However, it has many drawbacks, as described pre-
CD31 immunopositivity of sinusoidal endothelial cells viously. The current study aimed at encapsulating Sor into
(Fig. 15a & b and Fig. 16). Conversely, the liver of mice nano-emulsion followed by in vitro and in vivo compara-
treated with Sor-Susp revealed a significant increase in tive studies with Sor susp to tackle any improvement of its
CD31 immunoexpression compared to control and blank anticancer activity or safety due to its encapsulation into
mice (Fig. 15c and Fig. 16). However, sinusoidal endothe- nano-emulsion.
lial cells showed a significant reduction in CD31 immu- The formed nano-emulsion was characterized for particle
noreactivity in mice treated with Sor-NanoEm compared size zeta potential, and TEM determined its morphology.
to Sor-Susp (Fig. 15d and Fig. 16). The particle size of Sor NanoEm was significantly (P < 0.05)
Regarding the cerebral cortex blood capillaries of con- higher than the size of blank nano-emulsion, which might
trol and blank mice, there was mild CD31 immunopositiv- be due to drug encapsulation into the nano- droplet. This is
ity (Fig. 15e & f and Fig. 16). In contrast, cerebral cortex consistent with the literature, where Ifosfamide, a chemo-
blood capillaries expressed significantly elevated CD31 therapeutic agent encapsulated into Nano-Em, showed a
immunoreactivity in mice treated with Sor-Susp (Fig. 15g larger droplet size than the blank Nano-Em [51].
and Fig. 16). However, cerebral cortex blood capillaries of PDI value was ≤ 0.3. This indicated that the formed
mice treated with Sor-NanoEm showed a non-significant Nano-Em is monodisperse with a lower tendency of accu-
reduction in CD31 immunoreaction compared to Sor-Susp mulation and, therefore, could be suitable for drug delivery
(Fig. 15h and Fig. 16). application [51, 52, 52, 53]. Although the low negativity
of zeta potential was recorded, the Sor NanoEm was stable Sor release from Sor NanoEm was tracked by UV–Vis
with a low tendency of agglomeration as indicated by PDI spectrophotometric analysis. The analysis method was
value and TEM images; the low negativity might be attrib- validated by determining the accuracy and precision of
uted to Tween 80, a non-ionic surfactant, which was used inter-day and intra-day variations. The determined values
as an emulsifying agent for preparation of nano-emulsion. of precision indicated a high degree of repeatability and
Stability of nano-emulsion could be explained by the steric reproducibility. Following AOAC guidelines, the follow-
repulsive forces of Tween 80 (non-ionic surfactant) that ing equations, % RSD of repeatability = C −0.15 and %
are able to overcome the attractive van der Waals forces RSD of repeatability = 2 C −0.15, were used to calculate
and therefore, be able to stabilize nano-emulsion against the theoretical RSD% for intra-day and inter-day varia-
agglomeration [23, 54–56] tions, respectively, where C is the analyte concentration
TEM images showed nano-droplets with a spherical shape expressed as mass fraction. A valid method's accepted
with no signs of aggregation; however, the droplet size was practical %RSD ranged between ½ and 2 times the theo-
slightly smaller than the size recorded by Malvern Instru- retically calculated values. The obtained values matched
ment, and this might be due to different techniques applied, with what was recommended by AOAC guidelines, assur-
and this is consistent with the literature [50, 51, 57]. Both ing the validity of the UV–visible spectrophotometric
pH and viscosity values are acceptable for oral application. analysis method used in the current Study.
Fig. 13 a: l Immunohistochemically COX-2-stained liver, cerebral COX-2 immunoexpression in the cytoplasm of some hepatocytes and
cortex, and hippocampus sections.X400. a, e, i control mice and b, neurons (yellow arrows). d, h, l liver, cerebral cortex, and hippocam-
f, j blank mice liver, cerebral cortex, and hippocampus showed nega- pus, respectively, of sorafenib nano-emulsion showed mild COX2
tive COX-2 immunoexpression. c, g, k liver, cerebral cortex, and immunoexpression
hippocampus of sorafenib suspension-treated mice revealed positive
Fig. 14 A bar graph show-

ing the effect of sorafenib
powder suspended in 2.5%
CMC (Sor-Susp) and sorafenib
nano-emulsion (Sor-NanoEm)
on the area% covered by COX-2
positive immunoreactive cells
within the liver, cerebral cortex,
and hippocampus of mice.
Data are presented as mean
values ± SD. One-way ANOVA
is followed by Tukey's post hoc
test * Significant from the con-
trol groups, # Significant from
the blank group. x Significant
from Sor-Susp group, p < 0.05
Concerning the release study, Sor was ultimately released a sustained release behavior after 10 h (zero-order release
from its solution form after 8h, which is consistent with the kinetics). The fast release of Sor from Sor Nano-Em over
literature [58]. This is contrary to a lower release percent- the first 10 h might be explained by the large surface area
age recorded for Sor NanoEm simultaneously, followed by of the nano-droplet (the droplet size was 121.75 ± 12). This
Fig.15 a: h Immunohistochemically CD31-stained liver and cerebral (yellow arrow). e & f Control and blank mice's cerebral cortex blood
cortex sections.X400. a & b liver of control and blank mice showed capillaries had mild CD31 immunopositivity (yellow arrow). g The
mild CD31 immunoexpression of sinusoidal endothelial cells (yellow cerebral cortex blood capillaries of mice treated with sorafenib suspen-
arrow). c The liver of mice treated with sorafenib suspension showed sion showed moderate CD31 immunoreactivity (yellow arrow). h The
moderate CD31 immunoexpression (yellow arrow). d The liver of mice cerebral cortex blood capillaries of mice treated with sorafenib nano-
treated with sorafenib nano-emulsion had mild CD31 immunostaining emulsions showed mild CD31 immunostaining (yellow arrow)
prolonged action, fewer frequency of administration, and,

therefore, lower adverse events.
Chemotherapeutic medications are associated with toxic
adverse effects, including sickness behavior [60, 61]. The
underlying mechanisms of these adverse effects have yet
to be fully elucidated. In the present study, we investigated
some of the toxic effects of Sor therapy and the ability
of the new NanoEm formula to overcome these deleteri-
ous effects on the brain and livers of experimental mice
through direct action or via lowering the therapeutic effec-
tive dose needed. Sickness behaviors, including anxiety-like
behavior, depressive-like behavior, and cognitive deficits,
were investigated in addition to several biochemical and
Fig. 16 A bar graph showing the effect of sorafenib powder, sus- immunohistopathological analyses. Sor-Susp treated mice
pended in 2.5% CMC (Sor-Susp) and sorafenib nano-emulsion (Sor- showed a decrease in motor activity, a highly anxious state
NanoEm) on the area % covered by CD31 positive immunoreactive
portrayed by an increase in the dark chamber duration and
cells within the liver and cerebral cortex of experimental mice. Data
are presented as mean values ± SD. One-way ANOVA is followed by frequency associated with a reduction in the light chamber
Tukey's post hoc test * Significant from the control groups, # Sig- duration and frequency. Also, they displayed a depressive-
nificant from the blank group. x Significant from Sor-Susp group, like state evidenced by a decrease in the mobility duration
p < 0.05
and an increase in the immobility duration. These behavioral
changes were associated with a cognitive deficit represented
is consistent with a previous study [59], where linezolid, by a reduced SAP compared to control. These findings are
an antitubercular drug encapsulated into Nano-Em, showed in agreement with previous studies [42, 62–65] that inves-
zero-order release kinetics. tigated the sickness behavior associated with chemotherapy
Concerning the cytotoxicity study, Sor-NanoEm signifi- administration, including doxorubicin, cyclophosphamide,
cantly (P < 0.05) improved Sor's antiproliferative and cyto- and cisplatin. However, the administration of Sor Nano-Em
toxic effects on HepG2 cells. HepG2 cells were inhibited was able to reverse all these changes compared to Sor-Susp.
more efficiently with Sor Nano-Em than Sor-Susp, indicat- Growing evidence suggests that several potential medica-
ing that the nano-emulsion formula is more effective against tions, including Sor, displayed enhanced absorption and
HepG2 cells than Sor susp. Consequently, the required dose solubility after encapsulation into NPs [66]. Bearing in mind
of Sor is reduced with Nano-Em. This might lead to fewer side that the capability of the brain to take only up to 5% of the
effects and increase its efficacy, making Sor a safer and more initial NP dose, with rapid clearance from the circulation
effective chemotherapeutic drug for HCC treatment [60]. and a decrease in the blood–brain barrier crossing [67]. This
To ensure the safety of the new formula, a mouse-based could be the reason for the reduction of sickness behavior
LD 50 assay was carried out. First, the LD50 values of Sor associated with Sor susp administration.
Nano-Em administrated orally were estimated in mice accord- Histopathologically, the brain tissue of mice treated with
ing to procedures described previously [39]. Symptoms such Sor suspension displayed degenerative changes in the cer-
as severe diarrhea, paralysis, and seizures were observed in ebrum, hippocampus, and cerebellum histo-architecture.
the intoxicated mice. Different doses of Sor Nano-Em were This may be attributed to the passage of Sor through the
selected to analyze further changes in mice after intoxication blood–brain barrier. On the other hand, treatment with
quantitatively. It was found that the toxic effect of Sor Nano- Sor nano-emulsion resulted in less degenerative changes.
Em in mice was dose-dependent. Massive diarrhea and fluid Immunohistochemically, Sor susp treatment caused inflam-
loss in mice before death indicated that dehydration could mation and angiogenesis in the neuronal tissue of mice.
be one of the leading causes of the death of mice from Sor These results indicated several side effects associated with
Nano-Em overdose. Accordingly, 30 mg/kg was determined the medicinal application of Sor susp that might negatively
as a safe therapeutic dose to be applied in vivo. affect or limit its therapeutic application. Taken together,
Upon investigating the influence of the Nano-Em on Sor fewer side effects recorded with the application of Sor
pharmacokinetics, a comparative study was conducted ver- NanoEm on the brain revealed the merits of Sor encapsula-
sus Sor susp. The nano-emulsion significantly (P < 0.05) tion into nano-formulations.
increased the following pharmacokinetics parameters, Assessing the levels of hepatic enzymes is a com-
including Cmax, AUC, and t1/2 of Sor, along with reduc- mon way to determine the state of liver function. These
ing its elimination, clearance, and volume of distribution. enzymes leak into the bloodstream when hepatocytes are
This can ensure better drug characteristics with favorable damaged, and as a result, they serve as indicators of liver
damage. Increased levels of these biochemical variables anti-cancer efficacy. Additionally, Sor Nano-Em showed
could trigger carcinogenesis as they frequently corre- enhanced pharmacokinetic characteristics and reduced Sor
late with the prevalence of hepatotoxicity (Awad et al., dose with a lower incidence of side effects when admin-
2023). In this study, the elevated levels of ALT in the Sor- istrated in vivo. In vivo studies indicated that mice given
Susp group represented the progression of liver damage. Sor Nano-Em improved their behavioral profile and recov-
The findings showed that Sor Nano-Em administration ery of essential biomarkers, such as COX-2 inflammatory
improved liver function more than Sor-Susp therapy, dem- markers and liver enzymes. Notably, the structural integ-
onstrating the nano-emulsion formula's capacity to restore rity of the liver and brain returned to normal.
serum enzyme levels in hepatotoxic conditions by reduc- These results highlighted that encapsulation of Sor into
ing liver damage and boosting anti-cancer activity. Our nano-emulsion might be a potential alternative strategy for
results are consistent with recent research where scientists its application for better therapeutic management of car-
confirmed their agents' hepatoprotective and anti-cancer cinogenic patients compared to conventional Sor therapy.
properties by restoring normal levels of liver enzymes
[68–70]. Our results revealed increased alterations of
Author contributions Dalia Zaafar: Conceptualization, Methodol-
cytochrome P450 in Sor-Susp group samples, dramatically ogy, Formal analysis, Writing – original draft, Writing – review &
impacting metabolism, therapeutic efficacy, and adverse editing, Supervision. Heba M.A. Khalil: Methodology, Investiga-
drug reactions for itself and other drugs. Furthermore, tion, Methodology, Writing – review & editing. Gehad E. Elkhouly:
elevated CYP levels can induce liver injury through toxic Methodology, Formal analysis, Writing – original draft, Abanoub
Selim Sedeky: Methodology, Formal analysis, Writing – original
metabolites and reactive oxygen species production [71]. draft, Yasmine H. Ahmed: Conceptualization, Methodology, For-
The Sor-NanoEm restored normal levels of CYP, prevent- mal analysis, Writing – original draft, Mona G. Khalil: Conceptu-
ing the deleterious effect found in the suspended formula. alization, Methodology, Formal analysis, Writing – original draft,
In the current study, the histopathological investigations Yasmin Abo-zeid: Conceptualization, Resources, Methodology,
Formal analysis, Writing – original draft, Writing – review & edit-
revealed alterations in the hepatic tissue structure of mice ing, Supervision.
treated with Sor suspension, including dilated and congested
hepatic sinusoids with loss of hepatocyte standard structure. Funding Open access funding provided by The Science, Technology &
These findings agreed with Alkhatib and colleagues, who Innovation Funding Authority (STDF) in cooperation with The Egyp-
tian Knowledge Bank (EKB).
reported that mice treated with Sor revealed alterations in
hepatic tissue and dilated sinusoids [72]. Strumberg attributed Availability of data and materials Data will be made available from the
the hepatotoxic effect of Sor to its oxidative metabolism in the corresponding author upon request.
liver [73]. Immunohistochemical examination revealed severe
inflammatory lesions in the hepatic tissue of mice exposed to Declarations
Sor suspension, evidenced by significant intense COX2 expres- Ethics approval The experimental procedures adhered to the NIH
sion. These results align with the findings of Ting and col- Guidelines for the Care and Use of Laboratory Animals and were
leagues, who stated that rats injected with Sor showed severe approved by the Institutional Animal Care and Use Committee at the
inflammatory changes in hepatic tissue by H&E stain [74]. Faculty of Veterinary Medicine, Cairo University (Approval number:
Vet CU 09092023768).
Moreover, angiogenesis was observed in mice treated
with Sor suspension, evidenced by a significant increase Consent to participate N/A.
in CD31 immunoexpression in endothelial cells of hepatic
sinusoids compared to control mice. Otherwise, mice treated Consent for publication N/A.
with Sor-NanoEm revealed a substantially reduced angio- Competing interests The authors declare no competing interests.
genesis via decreased CD31 expression compared to the
Sor-suspension-treated group. Open Access This article is licensed under a Creative Commons
Conversely, mice treated with Sor Nano-Em revealed Attribution 4.0 International License, which permits use, sharing,
fewer degenerative, inflammatory, and angiogenesis changes adaptation, distribution and reproduction in any medium or format,
in hepatic tissue than those treated with Sor suspension. as long as you give appropriate credit to the original author(s) and the
source, provide a link to the Creative Commons licence, and indicate
if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless
Conclusion indicated otherwise in a credit line to the material. If material is not
included in the article’s Creative Commons licence and your intended
use is not permitted by statutory regulation or exceeds the permitted
The superior performance of the Sor-NanoEm formula- use, you will need to obtain permission directly from the copyright
tion in vitro, where it successfully suppressed HepG2 cell holder. To view a copy of this licence, visit http://creativecommons.
viability and induced apoptosis, demonstrated increased org/licenses/by/4.0/.
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Preparation and Characterization of Sorafenib Nano Emulsion: Impact On Pharmacokinetics and Toxicity An in Vitro and in Vivo Study

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Preparation and characterization of Sorafenib nano‑emulsion: impact

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Keywords Hepatocellular Carcinoma · Nano-emulsion · Sorafenib · hepG2 Cell line · Pharmacokinetics

Introduction These systems were also reported to overcome drug

Evaluation of Pharmacokinetic Parameters Behavioral study

Fig. 1 A flow chart showing

Fig. 2 TEM image of Sor-

Fig. 3 In vitro release profile of

% Cumulative Drug Release

Severe diarrhea accompanied by shivering was promptly

Pharmacokinetic Parameter Sor NanoEm Sor Susp

C max (µg/mL) 17.33 ± 0.24 9.6 ± 0.1

AUC0-t, area under the plasma concentration–time curve from zero

Depressive like behavior

As illustrated in Fig. 8a and b, the forced swim test indicated

Fig. 10 Effects of administer-

the liver and a non-significant reduction in neurons of Discussion

Fig. 14 A bar graph show-

prolonged action, fewer frequency of administration, and,

References Klebsiella pneumoniae Pneumonia-Septicemia Model in Rats.

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