1 s2.0 S2590156723000695 Main

International Journal of Pharmaceutics: X 7 (2024) 100225
Contents lists available at ScienceDirect
International Journal of Pharmaceutics: X

journal homepage: www.sciencedirect.com/journal/international-journal-of-pharmaceutics-x
Repurposing celecoxib for colorectal cancer targeting via pH-triggered

ultra-elastic nanovesicles: Pronounced efficacy through up-regulation of
Wnt/β-catenin pathway in DMH-induced tumorigenesis
Shahira F. El Menshawe a, Khaled Shalaby b, *, Mohammed H. Elkomy b, Heba M. Aboud a, *,
Yasmin M. Ahmed c, Abdelmeged A. Abdelmeged d, Marwa Elkarmalawy e, Mahmoud A. Abou
Alazayem d, Amani M. El Sisi a
a
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt
b
Department of Pharmaceutics, College of Pharmacy, Jouf University, Sakaka, Saudi Arabia
c
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Nahda University, Beni-Suef, Egypt
d
Department of Pharmaceutics, Faculty of Pharmacy, Nahda University, Beni-Suef, Egypt
e
Department of Pharmaceutics and Drug Manufacturing, Faculty of Pharmacy, Modern University for Technology and Information, Cairo, Egypt
A R T I C L E I N F O A B S T R A C T
Keywords: Celecoxib (CLX), a selective inhibitor for cyclooxygenase 2 (COX-2), has manifested potential activity against
Celecoxib diverse types of cancer. However, low bioavailability and cardiovascular side effects remain the major challenges
Colorectal cancer targeting that limit its exploitation. In this work, we developed ultra-elastic nanovesicles (UENVs) with pH-triggered
pH-triggered charge-reversal nanovesicles
surface charge reversal traits that could efficiently deliver CLX to colorectal segments for snowballed tumor
Box-Behnken design
Pharmacokinetics
targeting. CLX-UENVs were fabricated via a thin-film hydration approach. The impact of formulation factors
Wnt/β-catenin pathway (Span 80, Tween 80, and sonication time) on the nanovesicular features was evaluated using Box–Behnken
design, and the optimal formulation was computed. The optimum formulation was positively coated with pol
yethyleneimine (CLX-PEI-UENVs) and then coated with Eudragit S100 (CLX-ES-PEI-UENVs). The activity of the
optimized nano-cargo was explored in 1,2-dimethylhydrazine-induced colorectal cancer in Wistar rats. Levels of
COX-2, Wnt-2 and β-catenin were assessed in rats’ colon. The diameter of the optimized CLX-ES-PEI-UENVs
formulation was 253.62 nm, with a zeta potential of − 23.24 mV, 85.64% entrapment, and 87.20% cumula
tive release (24 h). ES coating hindered the rapid release of CLX under acidic milieu (stomach and early small
intestine) and showed extended release in the colon section. In colonic environments, the ES coating layer was
removed due to high pH, and the charge on the nanovesicular corona was shifted from negative to positive.
Besides, a pharmacokinetics study revealed that CLX-ES-PEI-UENVs had superior oral bioavailability by 2.13-fold
compared with CLX suspension. Collectively, these findings implied that CLX-ES-PEI-UENVs could be a prom
ising colorectal-targeted nanoplatform for effective tumor management through up-regulation of the Wnt/
β-catenin pathway.
1. Introduction Chemotherapy is the most important therapeutic option; nevertheless,

its long-term drug resistance offers considerable challenges (Srivastava
Colorectal cancer (CRC) remains a challenging ailment despite sub et al., 2021). In addition, the significant chemotherapeutic-related side
stantial treatment and early diagnosis breakthroughs, with poor overall effects might be deadly in certain cases. Therefore, the key to effectively
survival rates over the long term (Buzzelli et al., 2018). CRC is the treating this fatal disease is using therapeutic approaches with low or no
second most common cause of cancer-related deaths globally (Srivas toxicities. As a result, there is an imperious need for novel medications
tava et al., 2021). Smoking and diet (containing aromatic and hetero that can effectually treat CRC. Intriguingly, the approval of non-cancer
cyclic amines) are prime risk factors for CRC (Venkatesan et al., 2011). drugs has become a crucial strategy for accomplishing effective cancer
* Corresponding authors.
E-mail addresses: [email protected] (K. Shalaby), [email protected] (H.M. Aboud).
https://doi.org/10.1016/j.ijpx.2023.100225
Received 15 August 2023; Received in revised form 16 December 2023; Accepted 17 December 2023
Available online 20 December 2023
2590-1567/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
S.F. El Menshawe et al. International Journal of Pharmaceutics: X 7 (2024) 100225
therapies as a result of rapid drug development and reduced costs, as et al., 2015). These elastic cargos are constructed of Span 80 (surfactant)
information on pharmacokinetics, safety profiles, mechanisms of action, and Tween 80 as edge activator and exhibit great elasticity and
and formulations is already available to aid their clinical applications deformability due to the organization of surfactant macromolecules as a
(Hatem et al., 2019; Pushpakom et al., 2019). bilayer membrane completely enclosing an aqueous solute solution.
Evidence from numerous lines of research shows that CRC and Other hydrophilic surfactant substances increase the flexibility of lipid
adenomatous polyps have higher cyclooxygenase-2 (COX-2) as well as bilayers in spanlastic systems by creating pores and disrupting the
prostaglandin E2 levels (Wang and DuBois, 2006). Various cell culture, membranes (Tayel et al., 2015). During formulation, sorbitan-tailored
animal, and epidemiological investigations have established the anti- vesicles with flexible walls are produced by modifying traditional nio
tumorigenic potential of celecoxib (CLX), accomplished by selective somes with EA amalgamation. EA provides elasticity by destabilizing
COX-2 inhibition (Arber et al., 2006). In preclinical investigations, CLX vesicles and fluidizing their bilayers by decreasing interfacial tension (El
has shown significant anticancer effects against lung, colorectal, and Menshawe et al., 2019).
breast cancer (Hsiao et al., 2007; Bijman et al., 2008). Consequently, the The main obstacle to colon-targeted drug delivery is premature
FDA authorized CLX for the adjuvant therapy of individuals with CRC medication release in acidic environments (stomach and early small
(Steinbach et al., 2000; Kuwai et al., 2008). Actually, the expression of intestine) (Naeem et al., 2018). The surface features of nano-cargos have
COX-2 is activated by several mediators, including the Wnt/β-catenin been shown to substantially affect their selective accumulation inside
pathway, contrary to COX-1, which is constitutively synthesized in the cancer cells. According to a previous study, the cationic feature of DDS
tissues. Wnt proteins are a cluster of secreted ligands that regulate may facilitate mucoadhesion to CRC tissues and absorption by tumor
essential cellular functions, such as determining cell fates, cellular pro cells (Naeem et al., 2018). Therefore, using polyelectrolyte complexes in
liferation rates, survival, differentiation, and adhesion levels (Zhao synthesizing cationic UENVs could improve mucoadhesion, cellular
et al., 2022). The attachment of Wnt to its primary receptor stimulates absorption, and acidic stability. The use of positively charged poly
its signaling attracting the cytosolic protein (Kolluri and Ho, 2019) and ethyleneimine (PEI) assists in accumulating UENVs in tissues due to
interrupting the development of the axin/glycogen synthase kinase 3 interaction with negatively charged biological molecules (Naeem et al.,
(GSK3)/adenomatous polyposis coli (APC) complex and enhancing the 2018). In acidic environments, the cationic polymer coating may be
cytoplasmic accumulation of catenin (Terry et al., 2006), then trans incapable of preventing drug leakage. The Eudragit S100 (ES) coating
locating into the nucleus and interacting with T cell factor/lymph can resolve the issue of undesired rapid release in acidic conditions and
enhancer factor 1 (TCF/LEF1) to activate the production of Wnt- exhibit extended drug release in the colon. Therefore, coating the
targeted genes (Gehrke et al., 2009; Aoki et al., 2022). Similarly, the cationic UENVs with a pH-sensitive polymer such as ES could delay
stromal cell-derived factor 1/C-X-C motif chemokine receptor 4 (SDF-1/ medication release in the gastrointestinal tract (GIT). The layer would
CXCR4) axis signaling pathway is clinically significant for the metastasis disintegrate at a pH of 7.4 in the colon, exposing the cationic surface of
and spread of colorectal cancer (Kim et al., 2006; Wang et al., 2010). To UENVs and promoting their adsorption in malignant colon cells (Naeem
our knowledge, this work is the first to establish that invasiveness and et al., 2018). Hence, the proposed ES-PEI-UENVs could potentiate CLX
metastasis prevention in CRC occur through Wnt/β-catenin/TCF1/LEF1- bioavailability and protect against colon cancer.
mediated inhibition of COX-2. We hypothesized that inhibition of COX-2 Thus, in the current work, we formulated UENVs containing CLX
activity will decrease chemoresistance in CRC, suggesting a central (CLX-UENVs) by combining Span 80 and Tween 80 to be assessed as a
function for this system in such disease. possible therapy for colon cancer. The impact of formulation parameters
Despite the efficacy of CLX in CRC treatment, adverse effects, (Span 80, Tween 80, and sonication time) on UENVs features was
including thrombosis and cardiovascular risk, have limited its use in assessed using the Box-Behnken design, and the optimal formulation
cancer chemotherapy (Auman et al., 2008; Srivastava et al., 2021). To was estimated. The optimized CLX-UENVs formulation was positively
diminish the potential risk, it was recommended to use CLX for the coated with PEI (CLX-PEI-UENVs) and then coated with ES 100 (CLX-ES-
shortest feasible period at the lowest effective dosage. In addition, CLX PEI-UENVs). The designed CLX-ES-PEI-UENVs were evaluated physi
has limited water solubility and a narrow therapeutic index, making it ochemically to determine their suitability for colon administration. The
challenging to formulate with conventional dosage forms for the treat pharmacokinetics of CLX after oral administration of CLX-ES-PEI-UENVs
ment of CRC (Venkatesan et al., 2011). Furthermore, the therapeutic and an aqueous CLX suspension in rats were assessed to investigate the
dose of CLX in the tumor tissue was reported to be declined due to its efficacy of the developed systems. Furthermore, the underlying molec
rapid plasma elimination (Paulson et al., 2000). In addition, dimethyl ular mechanism for the antitumor activity of CLX on 1,2-dimethylhydra
sulfoxide (DMSO) is often used as a solvent for CLX during in vitro zine (DMH)-induced CRC in rats was explored.
studies, which is inappropriate for usage in vivo (Hsiao et al., 2007;
Sasaki et al., 2007; Bijman et al., 2008). Consequently, it is essential to 2. Materials and methods
identify a particular cancer cell-targeted drug delivery system (DDS) for
CLX administration. 2.1. Materials
The potency of an anticancer drug and the use of an efficient DDS
determine its efficacy in cancer patients. Thus, anticancer-targeted de Celecoxib was supplied from SEDICO Pharmaceutical Company
livery to the tumor site by modifying the biodistribution and pharma (Cairo, Egypt). Polyethyleneimine, Span 80, Eudragit S100, sodium
cokinetic features of DDS is a current focus of cancer therapy (Lu et al., carboxymethyl cellulose (MW: 90000 Da), 1,2-dimethylhydrazine,
2008; Hiremath et al., 2009). For cancer treatment, numerous DDS, methanol (HPLC grade), Tween 80, and chloroform (HPLC grade)
including polymeric micelles, parenteral emulsions, nanoparticles, and were purchased from Sigma-Aldrich (St. Louis, MO). Dialysis membrane
liposomes, have been explored (Mandal and Kundu, 2009; Qu et al., (MW cut off: 12000 Da) was purchased from SERVA Electrophoresis
2009; Kundu et al., 2010). Among these approaches, nanovesicular- GmbH (Heidelberg, Germany). Enzyme-linked immunosorbent assay
mediated transport offers several benefits, including a small diameter, (ELISA) kits for cyclooxygenase-2 (Catalog no. MBS266603) and Wnt-2
less toxicity, improved drug stability and solubility, greater medication (Catalog no. MBS7700340) were procured from MyBioSource (San
effectiveness, and the capacity to attain steady-state therapeutic levels Diego, CA, USA) while that for β-catenin (Catalog number K3383–100)
over a longer period (He et al., 2009). was obtained from BioVision (Waltham, MA, USA). Monoclonal anti
Among the promising nano-cargos for colon drug delivery, spanlastic body for CXCR-4 (Catalog no. PA1–21626) for immunohistochemistry
systems (novel ultra-elastic nanovesicles, UENVs) can be deemed as an assay and primers for TCF3 (Catalog no. PA5–20900) and LEF1 (Catalog
optimum approach. UENVs are surfactant-based colloidal drug carriers no. PA5102851) for qRT-PCR were purchased from ThermoFisher
with spheroid structures consisting of amphiphilic substances (Tayel (Inchinnan Business Park, Paisley, UK). Other ingredients and solvents
2
utilized were of analytical grade. allow them to mature, and then they were used for further experiments.
2.2. Design and optimization of experiments 2.4. CLX-UENVs characterization and optimization
Box-Behnken design is utilized for the generation of higher-order 2.4.1. Vesicle size (VS) and zeta potential (ZP) analysis
response surfaces, adopting lesser needed runs than a full factorial The mean CLX-UENVs diameter and polydispersity index (PDI) were
designing. It employed 12 middle edge nodes and three center nodes to assessed by the dynamic light scattering (DLS) technique in the Zetasizer
fit a second-order equation (Ahmad et al., 2020). Such design was used Nano ZS (Malvern Instruments, UK) (Aboud et al., 2018). Before anal
to study the independent variables effects on CLX-UENVs features using ysis, the CLX-UENVs dispersion was diluted with purified water (1:10),
Design Expert® software (12.0.3.0, Stat-Ease Inc., USA). Preliminary and the assessment was performed at room temperature (El Menshawe
experiments (data not presented) were conducted to establish the et al., 2019). The ZP of CLX-UENVs (surface charge) was assessed via a
probable independent variable ranges, which guided the choice of fac laser doppler micro-electrophoresis approach in Zetasizer Nano ZS.
tors and the used levels. Span 80 amount ranged from 160 to 200 mg,
Tween 80 amount ranged from 10 to 30% (w/w %), and sonication time 2.4.2. Measurement of entrapment efficiency
ranged from 0 to 10 min, were the independent variables. Vesicle size The EE of CLX in CLX-UENVs was determined by analyzing CLX in
(VS), zeta potential (ZP), entrapment efficiency (EE), and cumulative the supernatant and then deducting it from the amount added during
drug release after 24 h (CR) were the dependent variables. Fifteen formulation (10 mg). The supernatant containing free CLX was sepa
experimental runs (12 formulations and three repeated central points) rated using a cooling centrifuge (SIGMA 3-30 K, Germany) at 4 ◦ C
were conducted, as shown in Table 1. Using the R plot3D package, 3D (14,000 rpm, 2 h) (Khallaf et al., 2020). The resultant supernatant was
surface graphs were generated (Panda et al., 2021). The optimization then separated using a nylon syringe filter (0.45 μm). After appropriate
was based on a minimum VS and a maximum ZP, EE, and CR to get the dilution, the CLX concentration was estimated using a spectrophotom
formulation with the highest desirability (Salem et al., 2022). Table 1 eter (Shimadzu UV-1800, Tokyo, Japan) at 252 nm. The encapsulation
lists the independent variable levels and the experimental runs produced efficiency of CLX inside CLX-UENVs was estimated using the following
by the Box-Behnken design. formula:
Total amount of CLX − free CLX
EE% = × 100
2.3. Preparation of CLX-UENVs Total amount of CLX
CLX-UENVs were assembled via the thin film hydration procedure 2.4.3. Cumulative release (CR)
(El Menshawe et al., 2019). In this procedure, Span 80 and CLX (10 mg) The CLX release from CLX-UENVs was determined in vitro by vertical
were added to a round-bottom flask and dissolved by a chloroform- Franz cells (5 cm2) (Elkomy et al., 2022b). The release medium was
methanol combination (2:1). The organic solvent was then evaporated employed with pH values that gradually changed from 1.2 (acidic
at 55 ◦ C using a rotary evaporator (Heidolph Laborota 4000 Series, buffer, 2 h) to 6.8 (PBS, 6 h) and to 7.4 (PBS, 24 h), respectively, to
Heizbad, Germany) at 100 rpm, creating a thin film on the flask wall. By represent the stomach, upper small intestine and ileum, and colon
flask rotation in a water bath at 55 ◦ C and normal pressure for 30 min, (Vandamme et al., 2002). A certain quantity of various CLX-UENVs
the film was hydrated with phosphate-buffered saline (PBS, 10 mL, pH formulations (3 mg of CLX in each) was added to the donor chamber.
7.4) containing Tween 80. As seen in Table 1, the resultant dispersion Seventy mL of the release medium containing Tween 80 (0.1% w/v)
was then sonicated (Sonix TV ss-series, North Charleston, SC, USA). The were introduced to the receptor chamber. The experiments were con
formed dispersions were stored in a refrigerator (4 ◦ C) overnight to ducted at a temperature of 37 ◦ C and a stirring speed of 100 rpm. At
Table 1
Independent variables, experimental runs, and response variables for the formulations of CLX-UENVs according to the Box Behnken design.
Independent factors Levels
-1 0 1
X1: Span 80 (mg) 160 180 200

X2: Tween 80 (w/w %) 10 20 30
X3: Sonication time (min) 0 5 10
Run X1 X3 X2 VS (nm) ZP (mV) EE (%) CR (%) PDI¥
R1 160 10 5 267.81 ± 2.96 − 8.66 ± 0.18 50.03 ± 3.21 38.26 ± 5.06 0.157 ± 0.023
R2 160 20 0 264.43 ± 13.05 − 7.79 ± 0.68 68.62 ± 2.57 43.20 ± 3.64 0.457 ± 0.026
R3 160 20 10 232.17 ± 17.04 − 8.39 ± 0.49 60.21 ± 2.05 45.46 ± 3.89 0.086 ± 0.012
R4 160 30 5 227.62 ± 10.47 − 5.02 ± 0.20 58.26 ± 2.12 46.96 ± 3.26 0.115 ± 0.016
R5 180 10 0 219.18 ± 7.15 − 12.91 ± 0.13 83.48 ± 1.91 48.84 ± 4.13 0.142 ± 0.034
R6 180 10 10 217.74 ± 14.65 − 12.09 ± 0.40 76.67 ± 3.32 49.59 ± 3.24 0.397 ± 0.016
R7* 180 20 5 217.63 ± 4.12 − 11.85 ± 0.44 84.72 ± 2.73 59.36 ± 4.23 0.268 ± 0.021
R8* 180 20 5 213.86 ± 9.64 − 11.66 ± 0.47 85.35 ± 2.93 60.11 ± 2.93 0.214 ± 0.028
R9* 180 20 5 199.63 ± 9.45 − 10.43 ± 1.33 86.54 ± 1.97 62.74 ± 3.74 0.168 ± 0.019
R10 180 30 0 195.35 ± 18.77 − 9.51 ± 1.31 84.86 ± 2.04 63.86 ± 4.42 0.135 ± 0.023
R11 180 30 10 185.13 ± 26.70 − 9.09 ± 1.10 84.53 ± 3.39 67.62 ± 3.26 0.147 ± 0.015
R12 200 10 5 180.18 ± 25.25 − 13.83 ± 0.47 86.75 ± 2.72 72.88 ± 3.77 0.290 ± 0.026
R13 200 20 0 179.91 ± 24.16 − 13.06 ± 0.56 93.07 ± 2.57 82.65 ± 2.707 0.201 ± 0.026
R14 200 20 10 152.76 ± 15.33 − 13.27 ± 0.17 91.46 ± 2.29 90.16 ± 3.82 0.275 ± 0.023
R15 200 30 5 129.53 ± 10.85 − 12.81 ± 0.13 87.91 ± 2.51 93.92 ± 4.19 0.069 ± 0.033
Data are mean values (n = 3) ± SD.

*
Indicates the center point of the design.
¥
Excluded from optimization.
3
predefined intervals, aliquots (3 mL) were collected from the receptor optimized CLX-ES-PEI-UENVs. All formulations were delivered orally
chamber and replaced with an equivalent fresh receptor medium. The with adequate water. The animals had free access to food and drank
obtained aliquots were diluted, filtered, and subjected to spectropho until the evening before administration, after which they fasted for 12 h.
tometric analysis at 252 nm. The cumulative release (CR) results after Each rat was fed the oral suspension through stomach intubation, and
24 h were used in the optimization process. blood samples were collected at predetermined intervals via retro-
orbital puncture (1, 2, 3, 4, 6, 8, and 24 h post-dose). Blood was
2.5. Formulation of CLX-ES-PEI-UENVs immediately centrifuged (3000 rpm, 10 min) to separate plasma. After
collection, the plasma was immediately transferred to plastic tubes (5
The CLX-PEI-UENVs were fabricated by adding PEI (0.25%) to PBS mL) and preserved at 20 ◦ C till analysis.
containing Tween 80, and the same approach described above was fol
lowed. Furthermore, CLX-ES-PEI-UENVs were prepared by incubating 2.7.2. Chromatographic conditions
CLX-PEI-UENVs with moderate shaking for 1 h in an anionic solution of The quantitative analysis of CLX was determined using a slightly
ES in PBS at a core-to-coat ratio of 1:4 (Al-Mahallawi et al., 2017; Naeem modified LC-MS/MS method (Hu et al., 2021). The LC system consisted
et al., 2018). of Shimadzu Prominence (Kyoto, Japan) series with degasser (DGU-
20A3), an auto-sampler (SIL-20 AC) and a Zorbax C18 column (4.6 × 50
2.6. Characterization of CLX-ES-PEI-UENVs mm; 3.5 μm PS). The mobile phase comprised acetonitrile (80%) and
0.1% formic acid (20%). Ten μL was injected in LC-MS/MS with a flow
2.6.1. VS, PDI, ZP, and EE analysis rate of 1 mL/min. A negative mode was used for CLX and torsemide
As previously outlined, the VS, PDI, and ZP of CLX-ES-PEI-UENVs (internal standard). The autosampler and column temperatures were
were assessed. In addition, the EE of CLX inside CLX-ES-PEI-UENVs maintained at 25 ◦ C. Monitoring the transition of the m/z 380.125
was estimated. precursor ion to the m/z 316.100 for CLX and the m/z 347.196 pre
cursor ion to the m/z 262 for the torsemide was done in the multiple
2.6.2. Morphological evaluation reactions monitoring mode during ion detection. The ion spray voltage
The morphology of CLX-UENVs, CLX-PEI-UENVs, and CLX-ES-PEI- was established at 5500 V. The mass spectrometer used was AB Sciex
UENVs was assessed via a transmission electron microscope (JEM- Model (API 3200). Over the concentration range of 2–500 ng/mL, the
1400, Jeol, Tokyo, Japan). On a carbon-coated copper grid, one droplet calibration curve was linear (R2 = 0.999).
of the optimized formulation after dilution (1:10) was placed, allowed to
dry, and then stained with an aqueous solution of phosphotungstic acid 2.7.3. Samples preparation
(2%) as a negative stain. Finally, it is analyzed using a TEM with an 80 An aliquot of the internal standard (100 μL) in acetonitrile solution
kV (Salem et al., 2022). (100 ng/mL) was added to the plasma (0.5 mL) containing ethyl acetate
(4 mL). The mixture was vortexed, and the solvent was evaporated by a
2.6.3. pH-dependent in vitro drug release and ZP vacuum concentrator and then reconstituted with the mobile phase (0.5
As described before, vertical Franz cells were used to perform in vitro mL). The samples were then centrifuged (3000 rpm) for 10 min, and the
release tests of CLX from CLX-UENVs, CLX-PEI-UENVs, and CLX-ES-PEI- supernatant was injected into the LC-MS/MS system.
UENVs. The surface charge of CLX-ES-PEI-UENVs was measured by
incubating the dispersion in buffer solutions with varying pH levels (1.2, 2.7.4. Pharmacokinetic analysis
6.8, or 7.4) for a predetermined period while being shaken in a water The pharmacokinetics of CLX after oral administration of CLX-ES-
bath at 60 rpm and 37 ◦ C. Next, the resulting dispersion was centrifuged PEI-UENVs and conventional CLX suspension in rats were analyzed
and redistributed in distilled water. As mentioned before, the ZP of CLX- using a non-compartmental model (Aboud et al., 2020). The pharma
ES-PEI-UENVs was evaluated using a Zetasizer Nano-ZS. cokinetic parameters, including area under the curves (AUC), maximum
concentration (Cmax), half-life (t1/2), mean residence time (MRT), and
2.6.4. Short-term stability peak time (Tmax) were computed by the PK solver, an add-in program in
The stability study of the optimal CLX-ES-PEI-UENVs formulation Microsoft Excel.
was performed by keeping it in a glass vial in the refrigerator (4 ◦ C).
After 0, 1, 2, and 3 months, samples were drawn from the glass vial and 2.8. In vivo study of DMH-induced CRC in rats
analyzed for mean VS, EE, and ZP. In addition, the physical appearance
was evaluated for aggregation, separation, or precipitation. 2.8.1. Animals
The in vivo experimental model was implemented on adult male
2.7. In vivo pharmacokinetic studies Wistar rats procured from the animal house of Nahda University, Beni-
Suef, Egypt, weighing 200–250 g at the beginning of the protocol.
2.7.1. CLX administration to rats Rats were placed in an air-conditioned (25 ± 1 ◦ C) pathogen-controlled
The pharmacokinetics of the orally administered CLX-ES-PEI-UENVs animal room for seven days for adaptation before being subjected to
were compared with those of the orally administered conventional CLX laboratory experiments with free access to standard forage and tap water
suspension (CLX-SUSP). Following approval from the Local Institutional ad libitum. The experimental protocol for the animal study was
Animal Ethics Committee at Beni-Suef University (Acceptance no: approved by the Local Institutional Animal Ethics Committee at Beni-
022–292), twelve male Wistar rats (200–250 g), procured from the an Suef University (Approval no: 022–292).
imal facility of Nahda University (Beni-Suef, Egypt), were utilized in this
study. The procedures were conducted in accordance with the Guide for 2.8.2. Experimental design
the Care and Use of Laboratory Animals (2011) by the United States Rats were arbitrarily assigned into four weight-matched groups, each
National Academy of Sciences. After acclimatization, the rats were kept of 10 rats. The first group was a normal control and given only vehicles.
in propylene cages at room temperature. The rats were supplied with The second group (DMH-induced CRC) was kept as a positive control
ordinary food and had unlimited access to water. The animals were group and received only DMH (20 mg/kg/S.C) once for 12 weeks
arbitrarily split into two groups (n = 6). (Nascimento-Gonçalves et al., 2021). Group 3 received the DMH + CLX
The administered dose of CLX was 5 mg/kg (Bazan et al., 2016). The suspension. Group 4 received DMH + the optimal CLX-ES-PEI-UENVs;
first group was administered free CLX dispersed in sodium carbox CLX formulations were given in an oral daily dose after CRC induction
ymethyl cellulose (0.5%), while the second group was administered the within its therapeutic anti-inflammatory dose for rats (5 mg/kg body
4
Table 2 incubation with primary antibodies against CXCR 4 followed by incu

Primer sequences for the studied genes. bation with secondary antibodies and then with diaminobenzidine/
Gene symbol Primer sequence H2O2 as a chromogen. Slides were counterstained with hematoxylin
from 5′- 3′ then examined under a light microscope with the assistance of a
F: 5-TCCGTCGCCGGTCCACACCC-3 histopathologist.
β-actin
R: 5-TCACCAACTGGGACGATATG-3
F:5-CCAGACCAAACTGCTCATCCTG-3 2.9. Statistical analysis
TCF3
R: 5-TCGCCGTTTCAAACAGGCTGCT-3
F: 5-CTACCCATCCTCACTGTCAGTC-3
LEF1 The data were presented as mean ± SD. SPSS 22 (IBM SPSS Statistics,
R: 5-GGATGTTCCTGTTTGACCTGAGG-3
USA) was used to analyze the data using one-way ANOVA with a Tukey’s
post-hoc test for multiple comparisons. A p value <0.05 was regarded as
weight) once for 12 weeks (Bazan et al., 2016). Doses of test agents were significant. The western blot bands were quantified using the Image J
decided according to pilot study guided by the published art. At the end computer software (NIH, USA).
of this experiment, the animals were kept on overnight fasting and
sacrificed the following day using sterile instruments under anesthesia 3. Results and discussion
with 2.5% thiopental sodium (40 mg/kg, i.p.) before cervical dislocation
(Boztaş et al., 2021). 3.1. Experimental design and optimization
2.8.3. Tissue sampling The results of VS, ZP, PDI, EE, and CR of experimental runs with
Before cervical dislocation, rats were fasted for 24 h. The colon was various Span 80, Tween 80 concentrations, and sonication times are
entirely dissected and washed with saline. Colon was split into two shown in Table 1. The PDI values of CLX-UENVs ranged between 0.069
fragments; the first was preserved at − 80 ◦ C till the assays of COX-2, ± 0.033 and 0.457 ± 0.026 (Table 1). Low PDI values reflect a narrow
Wnt-2, β-catenin, and qRT-PCR analyses for TCF3 and LEF1; the sec size distribution and a homogeneous VS pattern, while high PDI values
ond portion was fixed in 10% formalin in normal saline for a minimum suggest more heterogeneity (Danaei et al., 2018). Analysis of PDI values
of 48 h until immunohistochemical assay of CXCR 4 and the histo using ANOVA Type III found insignificant effects on most independent
pathological assay. variables. Consequently, PDI was not included in the optimization step.
The wide variety of dependent variable findings suggests that vari
2.8.4. ELISA of tissue biomarkers ations in the levels of Span 80, Tween 80, and sonication time may
Levels of COX-2, Wnt-2 and β-catenin in colon tissue were deter significantly affect UENVs characteristics. Table 3 depicts the mathe
mined using ELISA test according to the sandwich technique described matical equations with coded values that best represent the causal and
earlier by Lequin (2005). Absorbance was measured by ELISA Process response factor interactions. The negligible lack of fit indicates that the
ing System (Model Spectra Max Plus-384 Absorbance Microplate models adequately described the observed variance (Table 3). Design-
Reader, USA). Expert calculated adequate precision to establish the dependability of
models used to traverse the design space (Eissa et al., 2022). Adequate
2.8.5. Determination of TCF3 and LEF1 expression levels using qRT-PCR precision in the statistical analysis of response variables was more than
Extraction of total RNA from the colon samples was executed using a 4, and predicted R2 values were close to adjusted R2 findings. In addi
Qiagen tissue extraction kit (Qiagen, USA) according to the manufac tion, the ANOVA analysis showed that changes in the levels of Span 80,
turer’s instructions. Total RNA yield was assessed using NanoDrop® ND- Tween 80, and sonication time significantly affected the response vari
8000 UV–Vis spectrophotometer (NanoDrop Technologies, Wilmington, ables. Fig. S1 (supplemental file) shows the diagnostic model plots that
DE, USA). According to the kit instruction, the extracted RNA was prove the appropriateness of the fitted models.
transcribed to cDNA using a high-capacity cDNA reverse transcription
kit (ferments, USA). qRT-PCR was implemented using the SYBR Green 3.1.1. Analysis of vesicle size (VS)
method. The content of 20 μL reaction mixture was 2 μL cDNA, 10 μL Actually, tumor cells create a neovasculature to guarantee a suffi
SYBR Green, 1 μL of specific primers (listed in Table 2), and 7 μL cient resource of nutrients and oxygen. As tumors enlarge, they recruit
nuclease-free water. The PCR conditions were carried out as follows: new vessels or engulf current blood vessels. Tumor vasculature is
denaturation at 95 ◦ C for 2 min; followed by 45 cycles at 95 ◦ C for 10 s, different from normal blood vessels as it is deficient of complete endo
59 ◦ C for 20 s, and 72 ◦ C for 30 s. β-actin was used for the normalization thelial lining with relatively large pores (0.1–3 μm in diameter) resulting
of the target genes data. Data analysis was performed by the 2-ΔΔCT in markedly greater vascular permeability. Nanoparticles are capable of
method to compute the relative expression level of the target gene (Livak penetration via the infirmly compacted vasculature to be delivered to
and Schmittgen, 2001). the tumor microenvironment and remain there due to the inadequate
lymphatic drainage of tumors. This phenomenon called enhanced
2.8.6. Histopathological study permeability and retention effect (EPR) which highlighted the possi
The prepared colon tissue slides were stained with standard Hema bility to utilize nano-sized drugs to passively target tumors (Nakamura
toxylin and Eosin (H&E) staining for the histopathological assay ac et al., 2016). Moreover, VS determines the cellular absorption, bio
cording to the method described by Banchroft and Gamble (Bancroft and distribution and circulation half-life of the nanosystem. Small-sized
Gamble, 2008). nanovesicles can be absorbed to a better extent than bigger ones due
to the fact that cellular uptake of the nano-cargo is size dependent
2.8.7. Immunohistochemical assay (Salem et al., 2022).
The immunohistochemical assay of tissue CXCR 4 was executed ac VS analysis was carried out to reinforce the nanoscale range of the
cording to technique designated earlier by Merz et al. (1995) through resultant nanovesicular dispersions. According to Table 1, the VS of the
5
Table 3
The outcomes of ANOVA analyses of response variables by the design expert software.
Source VS ZP EE CR
F-value p-value F-value p-value F p-value F p-value
Model 77.28 < 0.0001 38.98 < 0.0001 404.12 < 0.0001 1106.19 < 0.0001
X1: Span 80 190.21 < 0.0001 95.13 < 0.0001 2798.32 < 0.0001 8266.67 < 0.0001
X2: Tween 80 33.83 0.0001 21.79 0.0007 60.05 < 0.0001 1187.56 < 0.0001
X3: Sonication time 7.81 0.0174 0.0329 0.8593 56.12 < 0.0001 65.47 < 0.0001
X1. X2 20.07 < 0.0001 91.66 < 0.0001
X1. X3 16.08 0.0003 16.61 0.0003
X2. X3 13.43 0.0009 3.74 0.0618
X21 488.37 < 0.0001 229.46 < 0.0001
X22 165.40 < 0.0001 69.87 < 0.0001
X23 26.26 < 0.0001 1.73 0.1976
Lack of Fit 0.8665 0.6420 1.22 0.5287 13.46 0.0709 0.8236 0.6392
Model Linear Linear Quadratic Quadratic

Adjusted R2 0.9424 0.8906 0.9886 0.9958
R2 0.9547 0.9140 0.9910 0.9967
%CV 4.36 7.84 1.80 1.81
Predicted R2 0.9158 0.8419 0.9821 0.9943
Adequate precision 26.8490 19.7472 59.25 102.57
Standard deviation 8.95 0.8377 1.42 1.12
VS = 205.5 − 43.7.X1 − 18.4.X2 − 8.9.X3
ZP = 10.7 + 2.9.X1 − 1.4.X2 − 0.054.X3
EE = + 85.54 + 15.29X1 + 2.24X2 − 2.17X3 − 1.86X1 X2 + 1.64X1 X3 + 1.50X2 X3 − 9.40X12 − 5.47X22 + 2.18X32
CR = + 60.73 + 20.72X1 + 7.85X2 + 1.84X3 + 3.08X1 X2 + 1.31X1 X3 + 0.6229X2 X3 + 5.08X12 − 2.80X22 − 0.4410X32
developed nanovesicular formulations ranged from 129.53 ± 10.85 to 3.1.2. Analysis of zeta potential (ZP)
267.81 ± 2.96 nm. EAs-contained vesicles are typically spherical, have a ZP is the overall charge that vesicles have attained which can be
low propensity to agglomerate, and hence have small sizes (Aboud et al., considered as an indirect measurement for the judgment of colloidal
2020). Following transformation, the linear model was suggested for VS dispersions stability (Albash et al., 2019). High surface charge values
data. The influence of Span 80, Tween 80, and sonication time on the VS may prevent nanoparticles from aggregation by imbuing their surfaces
of CLX-UENVs formulations is shown in Fig. 1(A, B, and C). The VS with greater repulsion forces (Eid et al., 2021). The ZP values in Table 1
decreased as the level of Span 80, Tween 80, and sonication time ranged between − 5.02 ± 0.20 and − 13.83 ± 0.47 mV. Since all the
increased. formulations in this study had negative ZP values, variation in ZP will be
As illustrated in Fig. 1(A, B), VS was decreased while increasing the argued in terms of its absolute value to avoid misperception. It was
levels of Span 80. Indeed, the lower the HLB of the surfactant, the determined that the linear model was adequate for assessing the ZP data.
smaller the VS generated. The observed link between the size of nano ANOVA Type III-Partial Analysis demonstrated significant effects of
vesicles and HLB may be explained by reduction of surface energy Span 80 and Tween 80 on ZP values (p < 0.05), although sonication time
brought by increased hydrophobicity, resulting in smaller vesicles had no significant influence on ZP values.
(Khallaf et al., 2020). These findings were similar to those narrated by Of note, the ZP of vesicles reduced upon using Tweens instead of
Khallaf et al. (2020). Notably, EA (Tween 80) levels had a substantial Spans which might be assigned to the elevated HLB value of Tween 80
antagonistic effect on the mean VS. This may be interpreted as a (15) compared to Span 80 (4.3). As shown in Fig. 1(D, E), when the level
decrease in interfacial tension with increasing surfactant concentrations, of Span 80 was increased from 160 to 200 mg, ZP rose from |5.02| to |
resulting in the formation of small VS. In contrast, a low level of EA may 13.83| mV. This may result from the rising OH ion level, which is uti
be unable to cover the whole diameter of the vesicles. Therefore, vesicle lized to achieve high ZP (Abdelbari et al., 2021). In addition, the ZP
agglomeration may occur when the surface area is reduced to the point values declined gradually as the EA level increased which might be
that the EA may continue to exist throughout the whole surface of the resulted from the accumulation of hydrophilic EA on vesicular bilayers,
aggregation, creating bigger particles (Al-Mahallawi et al., 2017). resulting in the shielding of negative surface charges (Basha et al.,
Mahmoud et al. (2017) found comparable findings during the prepara 2013). Kim et al. (2000) claimed that the HLB value of the surfactant
tion of atorvastatin-loaded nanovesicular systems. As expected, impacts the competitive adsorption of OH ions at the interface that are
increasing the time of sonication decreased the VS, as shown in Fig. 1(B, existent in the hydration milieu. The lower HLB value of the surfactant
C). This may occur as a consequence of the exposure of the vesicles to (the nonpolar interface is more), the increased adsorbed OH which in
ultrasonic vibrations, leading to vesicle fragmentation (Elsherif et al., turn raises ZP. Moreover, the existence of (CH2-CH2-O)n in Tweens
2017). Such observations are supported by previous research findings created hydrogen bonds with water particles led to lower ZP values
(El-Say et al., 2016; El Menshawe et al., 2019). (Ibrahim et al., 2015). Our outcomes are concurred with those stated by
Albash et al. (2019).
6
Fig. 1. 3D plots for the impacts of independent variables on size (VS) and zeta potential (ZP) of CLX-UENVs.
Fig. 2. 3D plots for the impacts of independent variables on entrapment efficiency (EE) and cumulative release after 24 h (CR) of CLX-UENVs.
3.1.3. Analysis of entrapment efficiency (EE) independent factors substantially impacted the entrapment of CLX in
As elicited in Table 1, the developed CLX-UENVs formulations had side CLX-UENVs (p < 0.05). According to the regression results, Span 80
EE that ranged from 50.03 ± 3.21 to 93.07 ± 2.57%. The quadratic and Tween 80 disclosed a significant positive impact on the EE% results.
model was recommended for the analysis of entrapment data. The three In contrast, the encapsulation of CLX was negatively affected by the
7
sonication time. Higuchi kinetics elucidating a diffusion-controllable pattern, Table S1

As shown in Fig. 2(A-C), more than 90% of entrapment was obtained (supplemental file). Diverse reports disclosed that drug-loaded vesicular
when Span 80 level was more than 190 mg and Tween 80 at more than nanosystems would follow a controlled-release mechanism congruent
15%. The entrapment values increased when the levels of Span 80 were with Higuchi plots (Aboud et al., 2018; El Menshawe et al., 2019; Salem
raised which might be deduced based on the length of the alkyl chain, et al., 2022).
which affords high drug encapsulation (El Menshawe et al., 2019). In
addition, increasing the quantity of Span 80 (a vesicle-forming material) 3.2. Formulation optimization
may lead to the creation of a more lipophilic environment to house a
higher quantity of the hydrophobic drug (CLX), hence boosting the EE% The Design Expert® software made a number of recommendations
(Aziz et al., 2018). Concerning the influence of Tween 80, ANOVA that optimally fulfilled the specified constraints (minimum VS, and
analysis indicated that nanovesicles created with high Tween 80 levels maximum ZP, EE, and CR). The optimized formulation (X1: Span 80:
had a higher EE% than those prepared with low levels. The ability of EA, 200 mg, X2: Tween 80: 25.7% (w/w), and X3: Sonication time: 10 min)
when used optimally, to give more space for retaining more medications had a desirability value of 0.92. The experimental, expected, and pre
may explain the positive effect of EA on encapsulation (El Menshawe diction errors for the response variables of the optimal CLX-UENVs
et al., 2019). It may also be linked to the ability of the EA to create a formulation are manifested in Table 4. In addition, the computed per
stabilizing coating over the formed vesicles. Additionally, this coat may centage of prediction error for entire dependent variables was fewer
hold more drugs, raising the entrapment (Aboud et al., 2018). In than 11%. These results proved the validity of the ultimate models.
contrast, the fluidizing effect of EA at high amounts on the vesicle bi The standardized relative influences of the formulation parameters
layers may have led to the liberation of the entrapped drug (Eid et al., on the response variables are demonstrated in the Pareto chart (Fig. S2,
2019; Elkomy et al., 2022a; Elkomy et al., 2022b). supplemental file). VS, ZP, EE, and CR were more affected by Span 80
As shown in Table 3 and Fig. 2(B, C), it was also evident that the levels than by Tween 80 or sonication time.
sonication duration had an inverse marked impact on the entrapment, p
< 0.05. This may be a consequence of the rupture and reaggregation of 3.3. Characterization of CLX-ES-PEI-UENVs
vesicles, which results in micellar solubilization rather than entrapment
of the medication inside nanovesicles (El Menshawe et al., 2019). This 3.3.1. VS, PDI, ZP, and EE analysis
result is congruent with the results of Andersen et al. (2013) who The addition of PEI caused the VS to increase from 147.53 ± 11.44 to
showed a decline in entrapment as sonication duration increased during 176.81 ± 16.63 nm, and ZP shifted from – 15.40 ± 0.21 to +2.69 ±
the formation of chitosomes and pectosomes for metronidazole delivery. 0.09 mV. The inclusion of ES as a pH-sensitive layer altered the ZP from
positive (+ 2.69) to negative (− 23.24 ± 0.48 mV) and increased the VS
3.1.4. Analysis of cumulative release (CR) from 176.81 to 253.62 ± 18.45 nm, suggesting the successful produc
The cumulative release (CR) data after 24 h of all the assembled CLX- tion of CLX-ES-PEI-UENVs. ZP decreased after PEI surface coating,
UENVs formulations are presented in Table 1. The CR after 24 h ranged possibly due to the creation of a dense polymer layer that neutralized the
between 38.26 ± 5.06 and 93.92 ± 4.19%. The current study demon accumulated negative charge on CLX-UENVs. After surface coating with
strated that UENVs are ideal reservoirs for CLX. The quadratic model ES, the ZP exhibited negative ZP again due to the anionic nature of ES.
was recommended for the CR data analysis. From the regression co CLX-ES-PEI-UENVs depicted a PDI of 0.282, showing a narrow size
efficients, the levels of Span 80, Tween 80, and the time of sonication distribution. The EE of CLX within CLX-ES-PEI-UENVs (85.64 ± 3.64%)
significantly impacted CR values, p < 0.05. was slightly lower than that of CLX-UENVs (87.27 ± 3.06%) and CLX-
Concerning Span 80, it was observed that a high CR was produced PEI-UENVs (86.96 ± 4.57%). This might be attributed to a leak of the
when Span 80 levels were elevated (Fig. 2D and E). Rapid release at high encapsulated CLX during the ES layer addition procedure.
levels of Span 80 might be due to the existence of an unsaturated alkyl
chain, facilitating CLX leakage (El Menshawe et al., 2019). Furthermore, 3.3.2. Morphological evaluation
Span 80 had a low phase transition temperature (− 12 ◦ C), resulting in As elucidated in Fig. 3, the TEM revealed the morphology of CLX-
an increasing CR (El Menshawe et al., 2019). Notably, high Tween 80 UENVs, CLX-PEI-UENVs, and CLX-ES-PEI-UENVs. All UENVs formula
levels led to a high release rate. This is because Tween 80 has a short tions displayed spherical structures devoid of cracking and aggregation.
alkyl chain length, resulting in a fast release rate (Devaraj et al., 2002). Upon comparison of TEM micrographs and Zetasizer data, the average
Moreover, the reduced VS of UENVs with high levels of Tween 80 may VS via TEM micrographs was comparable to that recorded with DLS.
expose a larger surface area to the release environment, hence boosting
drug release. Additionally, CR data analysis showed that sonication had 3.3.3. pH-dependent drug release and ZP
a markedly positive impact on the CR of CLX-UENV formulations. The In the stomach condition, CLX-UENVs and CLX-PEI-UENVs demon
VS may assert this positive link between sonication time and CR. At any strated an early burst drug release (more than 40% after 2 h), followed
given moment, the fraction of the drug dissolved in the aqueous envi by an extended-release in the upper site of the small intestine condition,
ronment has an inverse correlation with the vesicle diameter. Thus, the as presented in Fig. 4. In the first 6 h, more than 65% of CLX was released
smaller VS might reduce the diffusional distance, thus accelerating drug from CLX-UENVs and CLX-PEI-UENVs. CLX-ES-PEI-UENVs, on the other
release rates (Wacker, 2013). hand, only released 18% of CLX in the first 6 h (6.4% in the stomach),
Kinetic analysis of CLX release data via linear regression elicited that showing that they could limit the CLX release in the upper GIT. The
the drug was released from the majority of the UENVs dispersions by release of CLX from CLX-ES-PEI-UENVs was much more rapid at the
terminal ileum (pH 7.4) than at acidic pH values. CLX-ES-PEI-UENVs
surface charge reversal property was evaluated in a milieu simulating
Table 4 the stomach, small intestine, colon and rectum pH conditions. In an
The results of dependent variables (software predicted, experimental, and pre
acidic environment, CLX-ES-PEI-UENVs retained a negative surface
diction error) of the optimized CLX-UENVs.
charge, which they changed to a positive superficial charge in a milieu
VS (nm) ZP (mV) EE% CR % (24 h) with a higher pH by dissolving the negatively charged ES layer.
Predicted 142.42 – 13.71 89.68 93.45 It’s interesting to note that CLX-ES-PEI-UENVs were designed for
Experimental 147.53 – 15.40 87.27 93.94 intelligent charge reversal (from negative to positive), which prevents
Prediction error (%)£ 3.46 10.97 – 2.76 0.52
undesirable mucus adhesion before reaching the colorectal segments,
£
(Experimental – Predicted) / Experimental x 100. restricts early burst release of CLX in the upper GIT, and then improves
8
Fig. 3. TEM morphology of CLX-UENVs (A), CLX-PEI-UENVs (B), and CLX-ES-PEI-UENVs (C).
Fig. 4. In vitro release profiles of CLX from CLX-UENVs, CLX-PEI-UENVs, and CLX-ES-PEI-UENVs.
the accumulation of nanovesicles and prolongs the release of CLX in the suspension and CLX-ES-PEI-UENVs administration to rats. Table 5 dis
target regions. The distinctive pH-triggered characteristic of surface plays the various pharmacokinetic characteristics of the two adminis
charge reversal would lead to greater CLX levels in the colorectal region, tered formulations after oral delivery. Following oral administration of
enhancing therapeutic efficacy (Niebel et al., 2012). CLX suspension, Cmax (480.51 ± 115.94) was reached in 3.17 ± 0.41 h;
however, the plasma levels of CLX dropped off quickly. Whereas
3.3.4. Short-term stability following administration of CLX-ES-PEI-UENVs, a comparatively slow
After storing for three months at 4 ◦ C, the optimized CLX-ES-PEI- rise and sustained plasma levels of CLX for a longer duration were
UENVs formulation had a milky appearance with no separation, aggre detected, with delayed Cmax (764.19 ± 23.91) occurring at 4.67 ± 0.94
gation, or precipitation. In addition, the changes in size, entrapment, or h, indicating an evidently regulated release of CLX from the CLX-ES-PEI-
surface charge are insignificant during the storage period, as illustrated UENVs. Additionally, the delay in mean Tmax from 3.17 h (CLX sus
in Fig. 5. The high stability of CLX-ES-PEI-UENVs may be attributed to pension) to 4.67 h (CLX-ES-PEI-UENVs) might be related to the capacity
their nanoscale range and high ZP (− 23.24). of ES to shield the entrapped CLX from the anticipated breakdown in the
stomach. After the fast stomach emptying of this aqueous dispersion, the
enteric coat will disintegrate quickly at the pH of the duodenum within
3.4. In vivo pharmacokinetic studies 15 to 30 min (Kendall et al., 2009; Tayel et al., 2015). These may be
assisted by the enormous surface area of the duodenum, the amorphous
Fig. 6 manifests the CLX plasma concentration-time curves after CLX
9
Fig. 5. The mean VS, EE, and ZP of the optimized CLX-ES-PEI-UENVs formulation after 0, 1, 2, and 3 months of storage in the refrigerator (4 ◦ C).
Fig. 6. CLX levels in rat plasma following oral administration of CLX suspension and CLX-ES-PEI-UENVs.
form of the entrapped medication, and the great dispersion level of the of CLX-ES-PEI-UENVs to carry drugs to the duodenal site of absorption,
drug (Tayel et al., 2015). offer transient drug protection from the acidic medium, and minimize
The prolongation in the MRT from 15.45 ± 1.99 to 18.98 ± 1.03 h as hepatic metabolism besides the permeation enhancing traits of the
well as in the elimination half-life from 11.06 ± 0.93 to 12.44 ± 0.55 h crafted nano-cargo. The enhanced bioavailability may help the CRC
for CLX suspension and CLX-ES-PEI-UENVs, respectively, could imply patient by lowering the therapeutic dose or increasing drug efficacy
the sustained release features of the fabricated nano-cargo. Depending (Tayel et al., 2015).
on the computed AUC0-24 values of CLX suspension (5068.40 ± 1577.32
ng.h/mL) and CLX-ES-PEI-UENVs (10,840.11 ± 790.29 ng.h/mL), the
CLX oral bioavailability was observed to be increased by approximately
2.13-fold. The improved oral bioavailability may be due to the potential
10
Table 5 MMP-9 and IL-1β (Zhang et al., 2019). Moreover, recent mesenchymal
Pharmacokinetic parameters of CLX after oral delivery of CLX suspension and stem cells (MSCs) studies suggest alternative CLX management for pa
CLX-ES-PEI-UENVs with the CLX dose of 5 mg/kg. tients with irritable bowel disease as a CRC precursor (Borowczak et al.,
Parameter CLX suspension CLX-ES-PEI-UENVs 2022). The MSCs migration towards CRC cells represents a wide sup
T1/2 (h) 11.06 ± 0.93 12.44 ± 0.55a
pression of immunological, inflammatory and apoptotic markers (Wang
K (h− 1) 0.0627 ± 0.0045 0.0557 ± 0.0024a et al., 2023).
Tmax (h) 3.17 ± 0.41 4.67 ± 0.94a Migration of MSCs to areas of tissue injury/inflammation has been
Cmax (ng/mL) 480.51 ± 115.94 764.19 ± 23.91a previously proven (Sadighparvar et al., 2020; Nascimento-Gonçalves
Mean Residence Time (h) 15.45 ± 1.99 18.98 ± 1.03a
et al., 2021). Inflammatory cytokines like interferon gamma (IFN-γ),
AUC0-24 (ng h/mL) 5068.40 ± 1577.32 10,840.11 ± 790.29a
AUC0-∞(ng h/mL) 6530.75 ± 2806.22 15,257.09 ± 1148.91a TNF-α, or IL-1, provoke MSCs at injury sites to express a wide variety of
Frel (%) – 213.88 immunosuppressive factors through their ability to release PGE2 (Zhang
et al., 2019). Indeed, in mouse models of colon carcinogenesis, the size
T1/2: terminal half-life; K: elimination rate constant; Tmax: time to reach Cmax;
Cmax: maximum drug concentration in plasma; MRT: mean residence time;
and quantity of adenomas are significantly reduced once COX-2 is
AUC0–24: area under plasma concentration–time curve from 0 to 24 h; AUC0–∞: inhibited, either pharmacologically or through genetic disruption
total area under plasma concentration–time curve; Frel: relative bioavailability. (Assoni et al., 2022). Alongside, it has been declared that signaling for
Listed data are mean values (n = 6) ± SD. TNF-α and IL-1β are two pathways that are typically dysregulated in
Using one-way ANOVA followed by Tukey’s post-hoc test. autoimmune disorders and cancer (Zhao et al., 2021). Besides, re
a
p < 0.05 versus oral CLX suspension. searchers have demonstrated that both signaling cascades directly
control arachidonic acid pathway and selectively stimulate the expres
3.5. In vivo study of DMH-induced CRC in experimental rats sion of PGE2-generating enzymes (Monteleone and Lutz, 2021). All
these data confirmed our results that CLX has potent anti-inflammatory
3.5.1. Effect of different CLX formulations on inflammatory markers and immunomodulatory effects, Table 6.
(COX-2 and IL-1β) levels
The average value results of rats exposed to DMH disclosed signifi 3.5.2. Effect of different CLX formulations on Wnt-2/β-catenin levels
cantly elevated inflammation signs in colon tissue homogenate levels of The Wnt-2/β-catenin pathway is strictly related to the incidence and
COX2 and IL-1β to 357.73% and 608.38%, respectively, compared to growth of tumors. Our data reveal that rats subjected to DMH showed an
normal control rats. Besides, treated groups received CLX suspension for aggressive downregulation in colon tissue mean value levels of Wnt-2/
12 weeks revealed a reduction in the intensity of inflammatory tissue β-catenin 1.27 ± 0.23/2.81 ± 0.32 by 21.42% and 36.12%, respectively
signs for COX2 and IL-1β by 58.02% and 26.87%, respectively compared compared to normal control rats. In contrast, CLX suspension exhibited
to DMH tissue levels. On the other hand, the treatment with CLX-ES-PEI- re-enhancement in Wnt-2/β-catenin tissue mean value levels to 2.89 ±
UENVs for 12 weeks markedly ameliorated both COX2 and IL-1β tissue 0.15/4.19 ± 0.24; while the group received 12 weeks of treatment with
levels compared to DMH induction group tissue levels, Table 6. CLX-ES-PEI-UENVs enhanced Wnt-2/β-catenin mean tissue levels more
Our results indicated the effect of CLX-ES-PEI-UENVs nanodispersion than DMH-induced CRC and CLX suspension levels to 3.76 ± 0.31/5.58
in the treatment of animals aggressive inflammation and immunological ± 0.35 (Table 6).
disorder from DMH-induced CRC. Previous data claimed that DMH Recent arts have displayed that Wnt-2/β-catenin is included in the
induced oxidative stress release coupled with apoptotic cells and various progression of numerous types of human carcinogenicity (Tewari et al.,
inflammatory markers including nuclear factor kappa (NF-κB), inter 2021; Hiremath et al., 2022). Researchers found that β-catenin mRNA
leukines (IL-6 and IL-1β) and COX-2 (Babu et al., 2023). Importantly, was much higher in frontline invasive CRC cells indicating its regulatory
arts have shown that CLX can block the expression and activation of NF- role in adherence junctions for hemophilic cell to cell adherence. In
κB and that it can fight off wide variety of molecules that contribute to addition, it is involved in cell signaling and gene transcription (Yuan
the expression, production, and secretion of inflammatory responses. et al., 2021). Indeed, the absence of Wnt-2 results in a lack of β-catenin
These molecules include tumor necrosis factor alpha (TNF-α), IL-1β, IL- and subsequently interferes with autophagy-promoting factors and
6, IL-8, COX-2 (Ferreira et al., 2021). A recent report has shown that the stimulates transcription of many signaling pathways involved in cell
selective overexpression of COX-2 in colon cancers has been related to proliferation (Lin et al., 2020) associated with many human diseases
the development of malignant tumors (Samadarsi et al., 2022). Such such as gastric, colon, and liver cancer (Flanagan et al., 2015). Many
observation suggests that inhibiting COX-2 will significantly decrease inflammatory tumors have demonstrated a cross talk among the in
the occurrence of CRC as mentioned in the American Cancer Society flammatory cascade and the Wnt/β-catenin signaling pathway (Tewari
reports on the rate of CRC in patients with inflammatory bowel ailment et al., 2021). In addition, microorganisms causing infections can cause
(Lee et al., 2021). Similarly, ovarian cancer model involved a great unchecked inflammation by overexpressing the Wnt/β-catenin pathway,
expression of COX-2, promoting the cancer metastasis via stimulating which can ultimately raise the risk of carcinogenesis (Anuja et al., 2017).
the phosphorylation of NF-κB, up-regulating the expression of C-myc In parallel, Wnt/β-catenin downregulation could stimulate the produc
and phosphorylated STAT, and snowballing the expression of MMP-2, tion of several inflammatory markers like COX-2 (Sobolewski et al.,
Table 6
Colonic COX-2, IL-1β, Wnt-2 and β-catenin levels in different studied groups.
Group COX-2 IL-1β Wnt-2 β-catenin
(pg/g tissue) (pg/g tissue) (pg/g tissue) (pg/g tissue)
Control 136.76 ± 2.53 43.37 ± 2.59 5.94 ± 0.23 7.78 ± 0.24

DMH 489.24 ± 20.09a 263.83 ± 26.67a 1.27 ± 0.15a 2.81 ± 0.32a
CLX suspension 283.86 ± 14.49a,b 70.90 ± 9.51b 2.89 ± 0.14a,b 4.19 ± 0.24a,b
CLX-ES-PEI-UENVs 181.18 ± 4.04b,c 60.77 ± 3.54b 3.77 ± 0.3a,b 5.58 ± 0.35a,b
Data are expressed as mean ± SD with n = 10 for each group.

Using one-way ANOVA followed by Tukey’s post hoc test.
a
Significantly differs from the control group.
b
significantly differs from the DMH group.
c
significantly differs from the CLX suspension group at p < 0.05.
11
Fig. 7. Colonic LEF 1 (A) and TCF 3 (B) gene expression levels in different studied groups. Data were expressed as mean ± SD with n = 10 for each group using one-
way ANOVA followed by Tukey’s post hoc test. a Significantly differs from the control group, b significantly differs from the DMH group, c significantly differs from
the CLX suspension group at p < 0.05.
2010), triggering overexpression of oxidative stress and numerous cy progression (Kaiser et al., 2023). Also, the cisplatin-induced ovarian
tokines production that enhance apoptotic factors and metastasis cancer model demonstrated that the down-regulation of LEF 1/TCF 3
(Nigam et al., 2023). Finally, our experimental results are congruent suppressed multicellular pathways related to ovarian cell carcinoma
with previous researches that targeted the effect of COX-2 inhibitors (Huang et al., 2023). Alongside, Zhou et al. (2017) exhibited that
towards the crosslink of upregulating the Wnt/β-catenin pathway. downregulation of MDR1/P-gp and inhibition of the Wnt/β-catenin
signaling pathway with miR-506 augmented CRC cell sensitivity to
3.5.3. Effect of different CLX formulations on LEF 1/TCF 3 gene expression oxaliplatin and induced upregulation of LEF 1/TCF 3 cascade. Prostate
The in vivo experimental LEF 1/TCF 3 gene expression results indi cancer model also manifested that β-catenin signaling promoted phos
cated that the DMH-induced CRC upregulated LEF 1/TCF 3 expression in phorylation of GSK-3 that engaged to the c-Myc promoter and interacted
colon tissues by 394.12% and 576.06%, respectively compared to with TCF4 independent of β-catenin resulting in increased cancer cell
normal control rats. In addition, the rat group received CLX suspension transcription (Schneider and Logan, 2018). Xia et al. (2010) reported
showed a significant improvement for LEF 1 expression by 72.83% and that treatment with CLX inhibited the β-catenin-dependent surviving
TCF 3 by 62.84% compared to DMH-induced CRC group. On the other activity of the osteosarcoma cell line. According to the previous
hand, treatment with CLX-ES-PEI-UENVs displayed a significant research, our results could be promising in the management of CRC by
improvement in LEF 1/TCF 3 gene expression compared to both CLX CLX via regulation of Wnt/β-catenin/LEF 1/TCF 3 expression levels
suspension and DMH-induced CRC groups (Fig. 7A, B). which is involved in disease progression and metastasis.
The T cell factor/lymphoid enhancer family genes encode for DNA
transcription factors via the SRY-related high mobility group (SOX) 3.5.4. Effect of different CLX formulations on CXCR4
domain (Liu et al., 2017). They have been involved in many diseases immunohistochemical staining
such as cancer (Peschel et al., 2022), and beta cells homeostasis (Locke Normal control group colon sections showed negative reactivity for
et al., 2011; Peschel et al., 2022). Additionally, they are involved in the CXCR4. The DMH CRC group showed an intense expression of CXCR4 in
Wnt signaling pathway (Takamoto et al., 2014). A model for breast the colon mucosa. CLX suspension treatment showed moderate positive
cancer manifested that upregulation of Wnt 3 induced a downregulation staining for CXCR4; while CLX-ES-PEI-UENVs showed a very mild to
of LEF 1/TCF 3 expression that inhibited cancer cells metastasis and limited expression of CXCR4 in the colon wall (Fig. 8A-D). Katoh et al.
12
Fig. 8. Imunopathological features of normal control group (A) showed no reactivity to CXCR4 in colon mucosa and wall; DMH group (B) showed strong reactivity
towards CXCR 4 in colon mucosal cells and colon wall; CLX- suspension (C) showed moderate colon mucosa and colon wall reactivity for CXCR 4; CLX-PEI-UENVs (D)
showed a very mild to limited expression of CXCR4 in colon wall.
(2010) observed that inhibition of COX-2 diminished CXCL12/CXCR4 On the other hand, treated CLX suspension colon sections revealed
expression besides expression of multiple other chemokines among lung moderate improvement. Most of the examined sections exhibited intact
cancer mice model. Moreover, CXCR7 was reported to alter the CXCL12 lamina epithelialis and propria with inflammatory edema occupying the
receptor that aided in tumor rabid transcription and metastasis (Katoh submucosa; while other sections showed mononuclear inflammatory
et al., 2010). Additionally, CXCR7 induced damage of CXCR4 provoking cells infiltration at the propria with goblet cells hyperplasia and hy
its inability to activate G proteins after CXCL12 binding (Xu et al., 2023). peractivity and other few sections showed complete destruction of the
Recently, Kassassir et al. (2023) stated that upregulation of CXCR4 is colonic wall with complete necrosis of mucosa and intense inflammatory
linked to cancer severity and survival. Bao et al. (2013) confirmed that cells infiltration, Fig. 9C. CLX-ES-PEI-UENVs exhibited better healing
the CXCL12/CXCR4 axis was involved in numerous pathways related to wherein part of the inspected sections disclosed obviously normal mu
carcinogenesis and played a crucial role in tumor development, survival, cosa with slight mononuclear inflammatory cells infiltration, Fig. 9D.
angiogenesis, metastasis, and resistance to treatment. Thus, our colon Recent research revealed that the histopathological mucosal damage has
section results of CXCR4 chemokines together with COX-2 inhibition via been healed upon treatment with CLX (Chen et al., 2023).
CLX-targeted nanoplatform could be employed as a successful thera
peutic drug delivery approach to tackle CRC. 4. Conclusions
3.5.5. Histopathological examination pH-triggered surface charge reversal nanovesicles, loaded with CLX,
Microscopic examination of colon tissue samples from the normal were successfully formulated to target the colorectal regions and
colon section group revealed a normal structure that encompassed inner augment the oral bioavailability of CLX. The formulated CLX-ES-PEI-
tunica mucosa containing glands with various mucus secreting cells and UENVs exhibited high stability. CLX-UENVs and CLX-PEI-UENVs
muscle in the outer layers, Fig. 9A. exhibited undesirable CLX release in a medium simulating the stom
Contrarily, DMH group sections exhibited marked histopathological ach and upper GIT pH. Nevertheless, the surface modification of CLX-
alterations; represented by a complete mucosal destruction with an PEI-UENVs with ES offered adequate protection for CLX in an acidic
intensive inflammatory reaction in all layers with presence of neutro milieu and enhanced CLX release in a medium simulating the pH of the
phils. Also, the glands displayed cystic dilatation with dysplastic alter colorectal regions. Consequently, the enhanced accumulation of CLX in
ation and a discernible decrease in goblet cells coupled with glandular the target segments was facilitated by pH-induced charge reversal of
epithelium exhibited hyperchromacia, anisokaryosis and frequent CLX-ES-PEI-UENVs. Moreover, oral administration of CLX-ES-PEI-
mitosis, Fig. 9B. Fleming et al. (2012) characterized a metastatic colo UENVs exhibited augmented bioavailability compared to CLX suspen
rectal histopathological model that indicates cystic dilatation with sion. In experimental rats, the tested CLX-ES-PEI-UENVs shielded
dysplastic changes coupled with neutrophils presence. Such outcomes against CRC induced by DMH and proved superior activity over con
coincide with Eisa et al. (2022) who reported the same pathological ventional CLX suspension. The apparent molecular mechanism behind
changes presented in our data results. this protection was via upregulation of Wnt/β-catenin pathway as well
13
Fig. 9. Photomicrograph (H&E) of colon represents normal control section (A) with higher magnification showed normal structure of colon glands with numerous
goblet cells; DMH group (B) showed cystically dilated gland (black arrow) with heavy inflammatory cells infiltration (red arrow), note the marked dysplasia in the
glandular epithelium (green arrow); CLX suspension (C) showed hyperplasia and hyperactivity of mucous glands (arrows); CLX-PEI-UENVs (D) showed apparently
normal propria with mild mononuclear inflammatory cells infiltration. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
as downregulation of LEF 1/TCF 3 gene expression and inflammatory Writing – original draft, Visualization, Validation, Software, Resources,
markers. Thus, the crafted CLX-ES-PEI-UENVs could confer a promising Methodology, Investigation, Formal analysis, Data curation. Amani M.
and tolerable nanoparadigm for CLX colorectal targeting which could El Sisi: Writing – review & editing, Writing – original draft, Visualiza
overwhelm its oral-related obstacles meanwhile accomplishing clinical tion, Validation, Supervision, Software, Resources, Project administra
benefits for CRC management. tion, Methodology, Investigation, Formal analysis, Data curation,
Conceptualization.
Funding
Declaration of Competing Interest
This work was funded by the Deanship of Scientific Research at Jouf
University under Grant Number (DSR-2021-01-03109). The authors wish to declare that no interest is involved in this
publication.
CRediT authorship contribution statement
Data availability
Shahira F. El Menshawe: Writing – review & editing, Visualization,
Validation, Supervision, Investigation, Conceptualization. Khaled Data will be made available on request.
Shalaby: Writing – review & editing, Validation, Supervision, Re
sources, Project administration. Mohammed H. Elkomy: Writing – re Appendix A. Supplementary data
view & editing, Writing – original draft, Visualization, Validation,
Resources, Investigation, Conceptualization. Heba M. Aboud: Writing – Supplementary data to this article can be found online at https://doi.
review & editing, Writing – original draft, Visualization, Validation, org/10.1016/j.ijpx.2023.100225.
Supervision, Software, Resources, Project administration, Methodology,
Investigation, Formal analysis, Data curation, Conceptualization. Yas References
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International Journal of Pharmaceutics: X 7 (2024) 100225

Contents lists available at ScienceDirect

International Journal of Pharmaceutics: X

Repurposing celecoxib for colorectal cancer targeting via pH-triggered

1. Introduction Chemotherapy is the most important therapeutic option; nevertheless,

X1: Span 80 (mg) 160 180 200

Run X1 X3 X2 VS (nm) ZP (mV) EE (%) CR (%) PDI¥

Data are mean values (n = 3) ± SD.

Table 2 incubation with primary antibodies against CXCR 4 followed by incu

F-value p-value F-value p-value F p-value F p-value

Model Linear Linear Quadratic Quadratic

VS = 205.5 − 43.7.X1 − 18.4.X2 − 8.9.X3

ZP = 10.7 + 2.9.X1 − 1.4.X2 − 0.054.X3

sonication time. Higuchi kinetics elucidating a diffusion-controllable pattern, Table S1

Control 136.76 ± 2.53 43.37 ± 2.59 5.94 ± 0.23 7.78 ± 0.24

Data are expressed as mean ± SD with n = 10 for each group.

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International Journal of Pharmaceutics: X 7 (2024) 100225

Contents lists available at ScienceDirect

International Journal of Pharmaceutics: X

Repurposing celecoxib for colorectal cancer targeting via pH-triggered

1. Introduction Chemotherapy is the most important therapeutic option; nevertheless,

X1: Span 80 (mg) 160 180 200

Run X1 X3 X2 VS (nm) ZP (mV) EE (%) CR (%) PDI¥

Data are mean values (n = 3) ± SD.

Table 2 incubation with primary antibodies against CXCR 4 followed by incu­

F-value p-value F-value p-value F p-value F p-value

Model Linear Linear Quadratic Quadratic

VS = 205.5 − 43.7.X1 − 18.4.X2 − 8.9.X3

ZP = 10.7 + 2.9.X1 − 1.4.X2 − 0.054.X3

sonication time. Higuchi kinetics elucidating a diffusion-controllable pattern, Table S1

Control 136.76 ± 2.53 43.37 ± 2.59 5.94 ± 0.23 7.78 ± 0.24

Data are expressed as mean ± SD with n = 10 for each group.

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Table 2 incubation with primary antibodies against CXCR 4 followed by incu