Adv Pharm Bull.

2025;15(2):371-389
doi: 10.34172/apb.025.43681 TUOMS
https://apb.tbzmed.ac.ir PRESS

Original Article

Codelivery of Raloxifene and Rutin as PEGylated

Nanoliposomes: Formulation, Characterization, and
Prophylactic Activity Against Breast Cancer
ID ID ID ID ID
Maryam Abdulmaged Oleiwi1 , Ali Al-Samydai1* , Aya Y. Al-Kabariti2 , Khaldun M. Al Azzam3 , Simone Carradori4 ,
Walhan Alshaer5 ID
1
Pharmacological and Diagnostic Research Centre, Faculty of Pharmacy, Al-Ahliyya Amman University, Amman 19328, Jordan
2
Department of Biopharmaceutics and Clinical Pharmacy, Faculty of Pharmacy, Al-Ahliyya Amman University, Amman
19328, Jordan
3
Department of Chemistry, Faculty of Science, The University of Jordan, 11942, Amman, Jordan
4
Dipartimento di Farmacia, Università degli Studi Gabriele d’Annunzio Chieti-Pescara, Via dei Vestini 31, Chieti 66100, Italy.
5
Cell Therapy Center, The University of Jordan, 11942, Amman, Jordan

Article info Abstract

Purpose: Breast cancer is the leading cause of cancer-related deaths among women. Chemotherapy
Article History: faces challenges such as systemic toxicity and multidrug resistance. Advances in nanotechnology
Received: August 23, 2024 have led researchers to develop safer and more efficient cancer treatment methods.
Revised: May 29, 2025
Methods: The thin-film hydration method was employed to synthesize PEGylated nanoliposomes
Accepted: June 4, 2025
epublished: June 28, 2025 (NLs) loaded with raloxifene (RLX) and a combination of RLX and rutin. The NLs were characterized
using a Zetasizer® instrument, transmission electron microscopy (TEM), and high-performance
Keywords: liquid chromatography (HPLC) analysis. The encapsulation of RLX and rutin was confirmed, and
Sustainability, Breast cancer, cell viability assays were conducted against breast cancer and normal endothelial cell lines.
PEGylated, Nanoliposomes, Results: The encapsulation efficiency significantly increased in the mixed formulation, with RLX
Cytotoxicity, HPLC
reaching 91.28% and rutin 78.12%, indicating successful encapsulation. These NLs remained
stable for up to two months at room temperature and one month at 4°C, demonstrating a biphasic
release pattern. After 24 hours, approximately 17% of RLX was released from the NLs and 25%
from the mixed NLs. In contrast, 55% of rutin was released from the NLs and 70.4% from the
mixed NLs within 72 hours. The inclusion of rutin or RLX in the liposomal formulation reduced
cytotoxicity against breast cancer cell lines, as indicated by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. However, it improved safety in normal human cells
and tissues.
Conclusion: PEGylated NLs loaded with RLX and rutin demonstrated safe anti-breast cancer
effects, outperforming mixed NLs, suggesting the potential for a safer and more targeted
treatment. Further investigations are needed into clinical translation.

Introduction tissue preservation and functional restoration through

Cancer is characterized by atypical cell proliferation and radiotherapy and imaging.4,5 Chemotherapy agents, such
dissemination, impacting life expectancy and healthcare as anthracyclines and platinum-based drugs, induce cell
systems.1 According to the World Health Organization, death by disrupting DNA strands. While customized
breast cancer (BC) is the most common and significant treatment plans are employed, side effects can be severe.⁶
cause of cancer-related deaths. Treatment for BC depends The cyclophosphamide + methotrexate + 5-fluorouracil
on factors such as disease stage, receptor status, and combination chemotherapy protocol helps reduce
patient preferences.2 It typically includes radiation therapy, recurrence.
chemotherapy, surgery, targeted therapy, immunotherapy, Strategies for lowering BC risk include avoiding
and hormonal therapy.3 tobacco, minimizing hormone therapy and radiation
Ionizing radiation generates electrically charged exposure, and maintaining a healthy weight. Research
particles, while chemotherapy targets cancer cells.4 has focused on personalized prevention strategies,
Surgical methods include tumor removal, mastectomy, precision medicine, immune system modulation, and the
lymph node dissection, and axillary lymphatic system tumor microenvironment.⁷ Raloxifene (RLX), a selective
removal. Current treatment approaches emphasize estrogen receptor modulator, is used to prevent and treat

*Corresponding Author: Ali Al-Samydai, Email: a.alsamydai@ammanu.edu.jo

© 2025 The Author (s). This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits
unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required
from the authors or the publishers.
Oleiwi et al

osteoporosis in postmenopausal women.8 drugs within NLs protects them from physiological
It reduces the risk of invasive bone marrow cancer but degradation, enhancing their activity while reducing
may not decrease the risk of noninvasive bone cancer. RLX exposure to healthy tissue. The efficiency of NLs depends
is contraindicated in patients with blood clots and it may on their physicochemical properties, including size
increase the risk of deep vein thrombosis and pulmonary and charge. The use of synthetic phospholipids, such as
embolism. Common adverse effects include hot flashes 1,2-dioleoyl-sn-glycero-3-phospho-L-serine, has been
and leg cramps.⁹ employed to improve liposomal activity by modulating
Anticancer therapies often lack selectivity, leading liposome structure and surface properties, generating
to adverse effects such as anemia and neuropathy.10 negatively charged NLs.23
Phytochemicals derived from plants exhibit anticancer Polyethylene glycol (PEG) is a highly hydrophilic and
properties and antioxidant activity. Flavonoids, biocompatible polymer known for its excellent solubility
glucosinolates, carotenoids, lignans, and resveratrol have in aqueous solutions, biocompatibility, and well-tolerated
been reported as potent antioxidants.¹¹ nature. The U.S. Food and Drug Administration (FDA)
Rutin (Figure 1), a bioactive compound found in citrus has approved PEG-conjugated pharmaceuticals for
fruits, apples, berries, and tea leaves, was first identified human use.24 PEGylation enhances material solubility but
in Ruta graveolens.12,13 Its pharmacological activities requires optimization for prolonged circulation. PEGs with
include managing Alzheimer’s disease, hyperkinetic molecular weights below 60 kDa tend to accumulate in the
movement disorders, and stroke, as well as preventing liver and lysosomes.25 The preparation of PEGylated NLs
neuroinflammation and promoting neural crest cell co-loaded with RLX and rutin may enhance selectivity,
survival.14 Rutin offers various health benefits, such as anticancer activity, and stability.
lowering hypertension, modulating blood coagulation, Rutin exhibits antiplatelet activity,26 whereas RLX’s
and preventing platelet aggregation.15-18 Additionally, it primary adverse effect is an increased risk of blood clot
improves hair and skin health, acts as a natural sunscreen, formation in the legs or lungs.27 Despite this risk, RLX is
supports atopic dermatitis management, enhances prescribed because its benefits are considered to outweigh
physical strength, and facilitates wound healing.19,20 its potential drawbacks, particularly for postmenopausal
Rutin, a potent antioxidant, has potential as an women at heightened risk of developing BC.28
anticancer drug due to its cytotoxic effects on cancer Combination therapy, which integrates
cells. These effects include inhibiting tumor growth, pharmaceuticals with dietary supplements and natural
preventing proliferation, and inducing cell cycle arrest.16 compounds, may yield comparable outcomes to
The antiangiogenic properties of rutin limit tumor access conventional chemotherapy but with fewer side effects.29
to oxygen and nutrients.17 Additionally, rutin causes DNA Traditional herbal therapies have demonstrated efficacy
damage in cancer cells, disrupting their genetic material in treating nasopharyngeal, breast, and pancreatic
and enhancing cytotoxicity. Its selective action minimizes cancers.30 Designing effective combination regimens
potential side effects while increasing its effectiveness in requires a thorough understanding of cancer biology and
cancer treatment. Combining rutin with conventional potential drug interactions. Research and clinical studies
treatments such as chemotherapy or radiation therapy indicate that combination therapy can improve cancer
may enhance its cytotoxic effects. However, further clinical treatment outcomes and survival rates.31-33
trials are needed to confirm its efficacy and safety.18,19 RLX, a selective estrogen receptor modulator, exhibits
Nanoliposomes (NLs) are small, spherical, or oval significant anticancer activity by binding to estrogen
structures composed of a phospholipid bilayer, forming receptors in mammary tissue, thereby inhibiting DNA
lipid vesicles ranging from 20 to 500 nanometers in size.20 transcription. It functions as a chemopreventive agent,
Due to their biodegradability, non-toxicity, and non- exerting estrogenic effects on bone, the cardiovascular
immunogenic properties, biocompatible materials serve system, breast tissue, and endometrium. RLX suppresses
as efficient carriers for various drugs.21,22 Encapsulating hormone-dependent BC cell proliferation, leading to
apoptosis and cell cycle arrest. Postmenopausal women at
elevated risk of BC may benefit from a five-year regimen
of 60 mg/day.34 In mouse models of triple-negative breast
cancer (TNBC), a daily oral dose of RLX inhibited tumor
growth, promoted regression, reduced epidermal growth
factor receptor (EGFR) expression, and diminished
tumorigenicity in human TNBC cells.35 Furthermore, the
combination of RLX and naringin increased antioxidant
activity, suggesting that co-delivery via nanostructured
lipid carriers could enhance therapeutic effectiveness and
reduce side effects.35
Figure 1. The chemical structure of rutin21 Molecular encapsulation within NLs is crucial for

372 Advanced Pharmaceutical Bulletin. 2025;15(2)

 Raloxifene and rutin as PEGylated Nanoliposomes

improving the stability and activity of pharmaceutical (Flawil, Switzerland). A UV–visible spectrophotometer
compounds. This method encapsulates active molecules (UV-1800) was obtained from Shimadzu (Kyoto, Japan).
within lipid bilayers, shielding them from enzymatic A vortex mixer and mini extruder were purchased from
degradation and harsh environmental conditions.36 VELP Scientifica (Velate MB, Italy), while a microscope
Park H proposed incorporating doxorubicin into NLs, was obtained from Nikon (Tokyo, Japan). A Nano Zetasizer
evaluating its efficacy using two distinct formulations: was purchased from Malvern (Cambridge, UK). The
Caelyx (pegylated liposomal doxorubicin hydrochloride) probe sonicator was acquired from BANDELIN (Berlin,
and Myocet (non-pegylated liposomal doxorubicin). Germany). An ELISA microplate reader was obtained
These formulations exhibited comparable anticancer from BioTek (Santa Clara, USA). A Shimadzu HPLC
efficacy with reduced cardiotoxicity.37 Additionally, NLs system (Prominence-i LC-2030C Plus, Kyoto, Japan) was
significantly enhanced the antiproliferative effects of LPSF used for analysis. The HPLC unit was equipped with a
by encapsulating inclusion complexes, thereby increasing UV-VIS Plus detector, a DGU-20A degasser, a SIL-20A
drug cytotoxicity.38-40 autosampler, and a solvent delivery system pump. The
This study aimed to investigate the anticancer and Chrom Quest software (version 4.2.34) was used to record
antioxidant properties of RLX and RLX-RUTIN-loaded signals on an LC-Solution workstation (version 1.25,
NLs against MCF-7, MDA-MB-231, and EA. hy926 cells, 2009–2010) (Shimadzu, Japan), running on Microsoft
with a focus on their selectivity. Furthermore, we explored Windows XP.
the effects of NLs on co-delivering RLX and rutin. This
research also developed a low-toxicity, BC-targeting NL Rutin determination using RP-HPLC
formulation of RLX loaded into PEGylated liposomes. Chromatographic conditions
Additionally, the impact of rutin on drug loading, The mobile phase consisted of a mixture of methanol and
liposome size, and stability was examined. water (1:1, v/v), adjusted to pH 2.8 with concentrated
phosphoric acid (85% w/w). The flow rate was set at 1
Materials and Methods mL/min, and the instrument operated in isocratic mode.
Materials The mobile phase was prepared daily, degassed in a bath
RLX was obtained from Carbosynth UK/International sonicator for 10 minutes, and filtered through a 0.45 μm
(New Delhi, India). Rutin was purchased from Sygnus filter paper before use. The column oven temperature
Biotech (Tokyo, Japan). Hydrogenated soybean was maintained at 40 °C, and separation was performed
phosphatidylcholine (HSPC) lipids, DSPE-PEG (2000) on a Fortis C18 column (150 mm × 4.6 mm, 5 µm) with
amine, and cholesterol were purchased from Avanti UV detection at 287 nm and 360 nm for RLX and rutin,
Polar Lipids (Alabama, USA). HPLC-grade chloroform respectively. The injection volume was 10 µL.
and methanol were purchased from Across Organics
(New Jersey, USA). Dulbecco’s phosphate-buffered saline Preparation of stock solution
(PBS) and Dulbecco’s modified eagle medium (DMEM) Approximately 2 mg of rutin was weighed and dissolved
were obtained from Euroclone SpA (Figino, Italy). PBS in 2 mL of methanol to obtain a 1 mg/mL solution. The
tablets and concentrated phosphoric acid (85% w/w) mixture was thoroughly vortexed, sonicated for 5 minutes,
were purchased from Fisher BioReagents (Pennsylvania, and then filtered through a 0.45 μm filter into HPLC vials
USA) and Sigma-Aldrich (Saint Louis, USA), respectively. for analysis using HPLC.41
Dimethyl sulfoxide (DMSO) and 70% alcohol were
obtained from Fisher Chemical (Waltham, USA). Standard solutions for calibration curves
The bromide (MTT) dye, Invitrogen To prepare the stock solution, 10 mg of rutin was
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium, dissolved in 10 mL of methanol. A series of dilutions
was purchased from Thermo Fisher Scientific (Waltham, was then prepared by taking 5.0 mL of the stock solution
USA). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was and diluting it with 5.0 mL of methanol, yielding a total
obtained from SRLchem (Maharashtra, India). Roswell volume of 10 mL. This resulted in standard solutions with
Park Memorial Institute (RPMI) medium was purchased rutin concentrations of 1000, 500, 250, 125, 62.5, 31.25,
from Euroclone SpA (Figino, Italy). and 15.625 µg/mL.

Instrumentation Determination of RLX using the RP-HPLC method

A digital balance (Ohaus Scales Adventurer) was used The same chromatographic conditions were indicated
for weighing (Parsippany, NJ 07054, USA). A digital pH above (chromatographic conditions).
meter was purchased from Jenway (London, UK), while
a centrifuge, microcentrifuge, CO₂ incubator, stirrer, Preparation of stock solution
sonicator, and water bath were acquired from Thermo Two milligrams of RLX were weighed and dissolved in
Scientific (Waltham, MA, USA). The Buchi Rotavapor 2 mL of methanol (1 mg/mL), thoroughly mixed using
R-300 and a freeze dryer were utilized throughout the study vortexing, and filtered through a 0.45 μm filter.

Advanced Pharmaceutical Bulletin. 2025;15(2) 373

Oleiwi et al

Standard solutions for calibration curves sonication at 35 °C for 10 minutes. The mixture was then
After weighing 10 mg of RLX and dissolving it in 10 mL centrifuged at 12,000 rpm for 10 minutes. The supernatant
of methanol, a final stock solution with a concentration was collected, filtered through a 0.45 μm syringe filter, and
of 1 mg/mL was obtained. Serial dilutions were then analyzed using HPLC.46
prepared at concentrations of 1000, 500, 250, 125, 62.5, Entraped drug
31.25, and 15.625 µg/mL. The solutions were mixed Encapsulation Efficiency
= ( EE% ) ×100%
Total drug
thoroughly, filtered through a 0.45 μm filter, and analyzed
to generate a calibration curve using Microsoft® Excel® The percentage of drug loading was calculated as
workbook software. A linear formula was derived, and the follows:
coefficient of determination (R²) was calculated and used
as a linearity parameter by ICH guidelines. Weight of loaded drug
Drug loading ( DL% )
= ×100%
Weight of lipids
Preparation of PEGylated NLs using the thin film
hydration method Characterization of the loaded NLs
In a round-bottom flask, lipids along with rutin and/or The NLs were characterized using dynamic light
RLX were accurately measured and dissolved in 5 mL of scattering (DLS) to determine their average size, PDI,
chloroform. To evaluate the impact of solvent variation, the and zeta potential. For analysis, each 50 μL sample was
results obtained using chloroform alone were compared diluted with 1 mL of deionized water. The same procedure
with those from a chloroform-methanol mixture in a 4:1 was followed for zeta potential measurement using a
% w/w ratio.42-45 All four NLs were prepared using the thin- zeta potential measuring cuvette. The zeta potential
film hydration method described by Al-Samydai et al.46 and particle size were analyzed using Zetasizer software
The specific quantities of lipids used for NL preparation provided by Malvern Instruments. All samples were tested
are listed in Table 1. in triplicate to ensure precision. To assess the thermal
The mixture was placed in a rotary evaporator at 50 °C stability of the formulation, the prepared NLs were stored
with an initial pressure of 350 mbar, which was gradually at room temperature and in a refrigerator at 4 °C for
reduced every 10 minutes until it reached 200 mbar. The two months.
process continued for 1 hour at a rotation speed of 70 rpm.
Afterward, the mixture was allowed to evaporate, forming In vitro drug release test
a thin film, and was then transferred to a -20 °C freezer for In vitro release testing was conducted using the NL
use the following day. formulation, pure rutin, and RLX solutions. The
The next day, the dried mixture was combined with a membrane was blocked in PBS for 24 hours to remove
PBS solution by vortexing for 30 minutes, followed by the preservative before use. One milliliter of RLX, rutin-
continuous heating in a hot water bath. This ensured mixed NLs, or a pure solution of rutin and RLX was placed
uniform suspension of all lipid components in the solution. into a dialysis tubing cellulose membrane. The membrane
The suspension was then incubated at 4 °C overnight to was washed with 10 mL of PBS (pH 7.4) at 37 ± 0.5 °C in
facilitate optimal lipid hydration.45,47 an aqueous bath under shaking at 100 rpm. One hundred
Subsequently, the NLs were extruded using a mini microliters of the release medium were removed at fixed
extruder. The extrusion process was repeated 13 times intervals (0.5, 1, 2, 4, 6, 24, 48, and 72 hours), replaced with
to ensure the NLs exhibited a low polydispersity index the same amount of prewarmed PBS, and then injected
(PDI). Unencapsulated compounds were removed by into the HPLC system to obtain the following equation:
centrifugation at 7000 rpm, and the supernatant was
Amount of drug released at time x
collected for further analysis following = the protocol Release ( % ) ×100%
Total amount of added drug
described by Al-Samydai et al.46
Lyophilization of liposomal formulations
Encapsulation efficiency and drug loading Following liposome extrusion, the samples were stored
The degradation of NLs was carried out by adding 800 at -70 °C for 24 hours, freeze-dried for an additional 24
µL of methanol to 200 µL of the NLs, followed by bath hours, and then refrigerated at 4 °C for one week. The NLs
were subsequently reconstituted in deionized water, and
Table 1. NLs formulations were prepared using the thin film method.
their stability was assessed using a Zetasizer.
Materials Free Formula 1 Formula 2 Formula 3

HSPC (wt %) 55 55 55 55 Morphological study

DSPE/PEG 2000 (wt %) 5 5 5 5 The morphology and structural configuration of the
Cholesterol (wt %) 40 40 40 40
mixed NLs were examined using transmission electron
microscopy (TEM). TEM imaging was performed using
RLX - 20 mg - 20 mg
the negative staining technique.48 Initially, 200-mesh
Rutin - - 20 mg 20 mg
Formvar copper grids from SPI Supplies (USA) were

374 Advanced Pharmaceutical Bulletin. 2025;15(2)

 Raloxifene and rutin as PEGylated Nanoliposomes

subjected to carbon coating via a low-vacuum Leica EM hours post-treatment).50

ACE200 glow discharge coating machine (Leica, Austria). The percentage of wound closure was calculated using
These carbon-coated grids were further treated with a the following formula:
1.5% solution of Vinylec K in chloroform. A droplet of the Area for day1 − Area for day 4
liposome suspension, diluted with deionized water, was Rate of wound closur ( % )
= ×100%
Area for day1
placed on the 200-mesh Formvar copper grid and allowed
to air dry. The prepared grids were then stained with a 3% In vitro antioxidant activity
(v/v) aqueous solution of uranyl acetate for 20 minutes at A DPPH solution was prepared by dissolving 2,2-diphenyl-
an ambient temperature. After incubation, the grids were 1-picrylhydrazyl in methanol to a final concentration of
rinsed with distilled water, air-dried, and subsequently 0.2 mM for the 96-well DPPH assay. Sample solutions
imaged using a Versa 3D TEM (FEI, Netherlands) containing RLX, rutin, and a physical mixture were
operated at 30 kV.46 prepared at varying concentrations using methanol as the
solvent. Stock solutions (2 mg/mL) were first prepared
Cell viability assay (MTT) for each component, followed by serial dilutions to
Two BC cell lines, estrogen receptor-positive (ER⁺) MCF- obtain seven different concentrations. Similarly, serial
7 and estrogen receptor-negative (ER⁻) MDA-MB-231, dilutions were performed for the nanoformulations. The
along with the normal endothelial cell line EA. hy926, DPPH solution was added to the wells, followed by the
were seeded into 96-well plates (1 × 10⁴ cells/well) and samples were added to their respective wells, including
incubated at 37 °C with 5% CO₂ for 24 hours. Rutin, RLX, blanks (methanol only) and vitamin C as the positive
and a combination of free and nanoliposomal formulations control. The microplate was incubated in the dark at
were applied in serial dilutions to determine the half- room temperature for 30 minutes. Absorbance was then
maximal inhibitory concentration (IC₅₀) using the MTT measured at approximately 517 nm using a microplate
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium reader. The percentage inhibition (I%) of the DPPH free
bromide) assay.49 radical was calculated using the following equation51:
The MTT assay measured the reduction of tetrazolium
Absorbance of sample
salt by mitochondrial dehydrogenases, producing a yellow Antioxidant activity ( % ) =
1− ×100%
Absorbance of control
tetrazolium compound proportional to the number
of metabolically active viable cells. After 72 hours of All tests were conducted three times, and the IC50 values
drug exposure, MTT was added, and mitochondrial are reported as the means ± SDs of triplicate samples.
activity was evaluated after four hours. To assess the
potential enhancement of RLX cytotoxicity within the Statistical analysis
nanoliposome formulation, cell proliferation was analyzed The results are expressed as the mean ± standard deviation
in formulations containing free RLX, rutin, mixed NLs, from a minimum of three separate trials. Significance was
and PEGylated NLs. assessed using various statistical tests (including paired
t-tests, one-way ANOVA, and multiple repeated measures
Migration assay ANOVA). A p value < 0.05 was considered to indicate a
The ER⁺ MCF-7 and ER⁻ MDA-MB-231 BC cell lines statistically significant difference. The analyses were
were plated in sterile 6-well culture plates at a density of conducted using SPSS software (Version 21, IBM Corp.),
800,000 cells per well and incubated at 37 °C with 5% CO₂ GraphPad Prism 6 (GraphPad Software Inc., USA), and
for 24 hours. The following day, a vertical scratch was Microsoft Office Excel (Microsoft, USA).
made at the center of each cell monolayer using a sterile
1,000 µL micropipette tip to simulate a wound for free Results and Discussion
drug and NL treatment. Each well was then rinsed twice The EE% was calculated, to ensure that only the
with sterile PBS. supernatant contained the drug, an indirect method
After 24 hours, cells were treated with RLX, RLX- was used by measuring the drug concentration in the
loaded liposomes (RLX Lipo), a physical mixture, or supernatant. The solubility of the drug in methanol under
mixed liposomes at a concentration of 0.5 × IC₅₀ or the experimental conditions was confirmed. A standard
IC₅₀ of the unencapsulated drug, as determined by the solution was prepared in HPLC-grade methanol at
MTT assay. Images of the wound areas were captured at the expected concentration for analysis. The solution
different time points using a phase-contrast microscope was visually inspected for any signs of precipitation or
(model P. MICRO-001, Nikon) with a 4 × magnification undissolved particles and was found to be completely
objective. The wound closure area was measured using clear, indicating full solubility.
Motic Images Plus version 2.0 software, with a reference Subsequently, the solution was analyzed using
closure distance of 2 µm. DMSO and untreated culture high-performance liquid chromatography (HPLC).
media served as negative controls. The wound closure rate Chromatographic analysis revealed a distinct and sharp
was assessed on day 1 (before treatment) and day 4 (72 peak corresponding to the drug, confirming its complete

Advanced Pharmaceutical Bulletin. 2025;15(2) 375

Oleiwi et al

dissolution in methanol without any solubility issues. This interference at the retention time corresponding to the
validation establishes methanol as a suitable solvent for analytical peak (Figure 2).
ensuring complete drug dissolution in the degradation
and analysis procedure. Linearity
The linearity range for the RLX calibration curves
HPLC analysis extended from 15.625 µg/mL to 1 mg/mL, with the
Qualitative and quantitative analysis of RLX curves plotted between peak area and concentration.
Various conditions were optimized during method The linear equation and correlation coefficient (R²) for
development to determine the most appropriate RLX were y = 2E + 07x + 66803 and 0.9995, respectively.
parameters for RLX analysis. Several wavelengths were The resulting linear regression equation demonstrates
tested, and to achieve high sensitivity, chromatographic a strong relationship between analyte concentration
separation was performed using an HPLC instrument and peak area (response). The method’s sensitivity is
(Shimadzu, Japan) with UV detection at a wavelength represented by the slope (2E + 07), indicating a significant
of 287 nm. The optimal mobile phase composition was response to concentration variations. The correlation
determined to be 40% PBS and 60% acetonitrile (ACN), coefficient (R² = 0.9995), being close to unity, signifies
delivered isocratically at a flow rate of 1 mL/min. A 10 µL excellent linearity within the examined range. This strong
injection volume was used to generate a sharp peak.1 R² value confirms the calibration curve’s close fit to the
experimental data with minimal deviation, reinforcing
Validation the accuracy and reliability of the analytical method for
System suitability parameters quantitative RLX determination.52
The stock solution was introduced into the
chromatographic system, and the system suitability Precision
parameters are presented in Table 2. In this method, the RSD was less than 2%, indicating that
the method has good repeatability, with a mean of 1.85%.
Specificity The low RSD value demonstrates that the method exhibits
The method demonstrated specificity since there was no excellent repeatability, ensuring it can reliably produce
comparable results across multiple trials. Precision is
Table 2. The system suitability for HPLC parameters.
crucial for ensuring reliability in quantitative studies,
No. Parameters RLX particularly for methods intended for routine quality
1 Retention time (tr) 2.392 control, where reproducible results are essential for
2 Theoretical plate (N) 2344 regulatory compliance and product safety.
3 Area (AUC) 1 mg/mL 13218933
RP-HPLC for rutin determination
4 Slope 2E + 07
The linearity range for the rutin calibration curves,
5 Intercept 66803 plotted between the peak area and concentration, was
6 Asymmetry (As) 1.34 from 15.625 µg/mL to 1 mg/mL. The linear correlation
7 LLOD 0.001 mg/mL coefficient for rutin was 0.9998. The absorbance was
8 LLOQ 0.015 mg/mL monitored at λmax = 360 nm. The linear equation and
RLX: Raloxifene; LLOD: The lower limit of detectionl; LLOQ: The lower limit correlation coefficient (R²) for rutin were y = 1E + 07x -
of quantification. 30539 and 0.9998, respectively. The method demonstrated

Figure 2. HPLC chromatogram for the standard injection of RLX (1 mg/mL). The mobile phase was as follows: methanol: water ratio of 1:1 (v/v), pH 2.8 (using
concentrated phosphoric acid (85% w/w)); flow rate: 1 mL min−1 (isocratic mode); column oven: 40°C; column: Fortis C18 (150 mm × 4.6 mm, 5 µm); wavelength
detection: 287 nm; and injection volume: 10 µL

376 Advanced Pharmaceutical Bulletin. 2025;15(2)

 Raloxifene and rutin as PEGylated Nanoliposomes

specificity, as the retention time of the analytical peak Levene’s test (P = 0.114). Further analysis of the different
remained unaffected by any interference (Figure 3). groups revealed significant differences, with a p-value of
P ≤ 0.001, as shown in Table 3.
Effect of solvent and rutin on encapsulation efficiency
(EE%) Effect of rutin on the encapsulation efficiency of RLX
Evaluation of the effect of the solvent on the EE% of RLX To assess the influence of rutin in the preparation
To assess the influence of the solvent used in the preparation process, its addition to the formulation of RLX led to
process, adding methanol to the mixed formulation F3 a significant increase in the EE of RLX, from 51.97
resulted in a significant increase in the EE% of RLX, (standard deviation: 2.3) to 91.28 (standard deviation:
from 63.86 (standard deviation: 8.81) to 91.28 (standard 0.07). This enhancement highlights rutin’s essential role
deviation: 0.07). The data revealed unequal variances, as in optimizing the encapsulation process. The substantial
indicated by Levene’s test (P = 0.035). Further analysis of increase in EE suggests that rutin may contribute to
the different groups revealed significant differences, with stabilizing RLX within the encapsulating matrix, possibly
a p-value of 0.033, as shown in Table 3. due to its antioxidant properties or its influence on the
There is a statistically significant difference (P < 0.05) structural integrity of the delivery system.
between the mixed formulation with and without The data indicated unequal variances, as evidenced
methanol, as indicated by the p-value of 0.033. by Levene’s test p-value of 0.032. Further analysis of the
However, although this difference is noteworthy, the different groups revealed significant differences, with a
p-value for RLX formulations is 0.114, indicating that this P value of 0.001, as shown in Table 4, confirming rutin’s
difference it is not statistically significant at the 0.05 level. positive effect on EE.
To assess the influence of the solvent used in the The difference in EE between formulations with and
preparation process incorporating methanol into the without rutin is statistically significant, as indicated by
formulation significantly improved in its EE, from 13.01 the highly significant P value (0.001) (P < 0.05). This
(standard deviation: 0.08) to 51.97 (standard deviation: demonstrates that rutin is a crucial component of the
2.31). This substantial improvement highlights the critical formulation, exerting a noticeable and meaningful effect
role of solvent selection in optimizing the encapsulation in enhancing EE%.
process and enhancing drug loading efficiency. Methanol
likely improved RLX solubilization and interactions Effect of solvent on the EE% of rutin alone
with the encapsulating material, thereby increasing To assess the influence of methanol addition on the
overall drug entrapment within the formulation matrix. EE% of rutin alone, the incorporation of methanol into
The data indicated equal variances, as demonstrated by the formulation resulted in a significant increase in the
Table 3. The effect of the solvent on the encapsulation efficiency (EE%) of RLX was assessed.

Formulation Solvent Mean Standard deviation Sig. (2-tailed)

Mixed With methanol 91.28 0.073

0.033
Mixed Without methanol 63.86 8.811
EE%
RLX With methanol 51.97 2.410
0.114
RLX Without methanol 13.01 0.080
RLX: Raloxifene; Mixed (Raloxifene and rutin).

Figure 3. HPLC chromatogram for the standard injection of rutin (0.5 mg/mL). The mobile phase was as follows: methanol: water ratio of 1:1 (v/v), pH 2.8 (using
concentrated phosphoric acid (85% w/w)); flow rate: 1 mL min−1 (isocratic mode); column oven: 40°C; column: Fortis C18 (150 mm × 4.6 mm, 5 µm); wavelength
detection: 360 nm; injection volume:10 µL

Advanced Pharmaceutical Bulletin. 2025;15(2) 377

Oleiwi et al

EE% of rutin, rising from 67.84 (standard deviation: and further analysis confirmed significant differences
0.045) to 78.12 (standard deviation: 0.39). The data between groups, with a P value of ≤ 0.001 (Table 7). Since
indicated unequal variances, as evidenced by Levene’s this P value is highly significant (P < 0.05), the observed
test (P = 0.006). Further analysis of the different groups reduction in EE% is unlikely due to chance. This finding
revealed significant differences, with a P value ≤ 0.001, as highlights RLX as a key factor in lowering encapsulation
shown in Table 5. The observed difference is statistically efficiency within this formulation.
significant and unlikely to be due to chance, as indicated
by the highly significant P value ( ≤ 0.001). These findings Nanoformulation characterization
demonstrate that methanol plays a crucial role in Characterization of the particle size, PDI, and charge of NLs
enhancing encapsulation efficiency. The average size, PDI, and charge of the freshly prepared
NLs were assessed, with measurements taken in triplicate
Effect of solvent on the EE% of rutin in the mixed formulation for each run.
To evaluate the impact of methanol on the EE% of rutin This study evaluated the impact of loading materials on
in the mixed formulation, its effect was analyzed during nanoparticle characterization. The results showed that the
the preparation process. The addition of methanol led to particle size of mixed-loaded NLs (F3) was 125.38 nm,
a significant reduction in EE%, decreasing from 38.11% RLX-loaded NLs (F1) 138.25 nm, and free NLs 123.56
(SD: 0.12) to 21.03% (SD: 0.98). This decline suggests nm. ANOVA, followed by the least significant difference
that methanol disrupts the encapsulation process under (LSD) test, revealed significant differences among the
the given conditions. Its solvent properties may interfere groups (P < 0.05), as shown in Table 8. However, no
with rutin’s interaction with the encapsulating material, significant difference was observed between co-loaded
potentially altering solubility, weakening hydrophobic NLs and free NLs (P = 0.567). In contrast, RLX-loaded
interactions, or inducing premature drug leakage. NLs showed significantly larger particle sizes compared
Levene’s test confirmed unequal variances (P = 0.002), to both co-loaded and free NLs (P < 0.05). Notably, all
and further statistical analysis revealed a highly significant formulations remained within the optimal size range
difference between the groups (P ≤ 0.001), as presented for NLs ( < 300 nm), ensuring their suitability for drug
in Table 6. These findings indicate that methanol delivery applications.
adversely affects rutin encapsulation efficiency in mixed The PDI values were 0.1237 for mixed-loaded NLs,
formulations. The strong statistical significance (P = 0.001, 0.1408 for RLX-loaded NLs, and 0.1930 for free NLs.
P < 0.05) confirms a notable difference in EE% between ANOVA and LSD testing indicated significant differences
the conditions, reinforcing methanol’s detrimental impact among the groups (P < 0.05). While mixed-loaded and
on encapsulation efficiency in this formulation. RLX-loaded NLs showed no significant difference
(P = 0.205), free NLs significantly differed from both
Effect of mixture formation on the EE% of rutin in the (P < 0.05). Importantly, all formulations maintained a PDI
mixed formulation below 0.300, confirming homogeneous size distribution
To assess the impact of RLX on the EE% of rutin during and enhanced stability. The lower PDI of mixed-loaded
the preparation process, RLX was incorporated into the NLs suggests improved uniformity when RLX and rutin
formulation. This addition led to a significant reduction are combined, as presented in Table 8.
in EE%, decreasing from 78.12 (SD: 0.39789) to 38.11 (SD: The zeta potential, a crucial indicator of surface charge
0.121). The substantial decline suggests that RLX interacts and stability, measured -10.7 mV for mixed-loaded NLs,
with the encapsulating matrix or competes with rutin for -4.2 mV for RLX-loaded NLs, and -5.04 mV for free NLs.
entrapment sites, thereby reducing the system’s capacity to ANOVA and LSD testing revealed significant differences
retain both compounds effectively. among the groups (P < 0.05), except between free NLs and
Levene’s test indicated unequal variances (P = 0.015), RLX-loaded NLs (P = 0.454). Co-loaded NLs exhibited
Table 4. Effect of rutin on the encapsulation efficiency of RLX Table 6. Effect of solvent on rutin’s encapsulation efficiency (EE%) in the
mixed (raloxifene and rutin) formulation.
Formulation Mean Standard deviation Sig. (2-tailed)
Formulation Mean Standard deviation Sig. (2-tailed)
Encapsulation With Rutin 91.29 0.073
Efficiency 0.001 Without Methanol 38.11 0.121
EE% 0.001
(EE%) Without Rutin 51.98 2.317 With Methanol 21.03 0.987

Table 5. Impact of solvent on the encapsulation efficiency (EE%) of rutin Table 7. Effect of the mixture on the encapsulation efficiency (EE%) of rutin in
alone. the mixed (raloxifene and rutin) formulation.

Formulation Mean Standard deviation Sig. (2-tailed) Formulation Mean Standard deviation Sig. (2-tailed)

Without Methanol 67.84 0.045 Without RLX 78.12 0.398

EE% P ≤ 0.001
EE% ≤ 0.001 With RLX 38.11 0.121
With Methanol 78.12 0.398
RLX: Raloxifene.

378 Advanced Pharmaceutical Bulletin. 2025;15(2)

 Raloxifene and rutin as PEGylated Nanoliposomes

Table 8. Influence of loading on the characterization of NLs

Standard ANOVA
Parameter NLs Mean Multiple Comparisons LSD
Deviation F Sig.

Co Loaded NLs 125.38 1.51 Co Loaded NLs RLX Loaded Nanoliposomes P ≤ 0.001

Size (nm) RLX Loaded Nanoliposomes 138.25 6.51 17.32 P ≤ 0.001 - Free Nanoliposomes 0.567

Free Nanoliposomes 123.57 1.59 RLX Loaded Nanoliposomes Free Nanoliposomes P ≤ 0.001

Co Loaded NLs 0.124 0.020 12.76 0.002 Co Loaded NLs RLX Loaded Nanoliposomes 0.205

PDI RLX Loaded Nanoliposomes 0.141 0.024 - Free Nanoliposomes 0.001

12.76 0.002
Free Nanoliposomes 0.193 0.006 RLX Loaded Nanoliposomes Free Nanoliposomes 0.006

Co Loaded NLs -10.75 1.13 33.52 P ≤ 0.001 Co Loaded NLs RLX Loaded Nanoliposomes P ≤ 0.001
Zeta
potential RLX Loaded Nanoliposomes -4.25 1.90 33.52 P ≤ 0.001 - Free Nanoliposomes P ≤ 0.001
(mV)
Free Nanoliposomes -5.04 0.650 33.52 P ≤ 0.001 RLX Loaded Nanoliposomes Free Nanoliposomes 0.454
PDI: The polydispersity index; RLX: Raloxifene; NLs: Nanoliposomes; LSD: Fisher's least significant difference;
F: F-Statistic.

a significantly greater negative charge than the other a

450
formulations (P < 0.05). All formulations-maintained zeta 400
potential values within the optimal range (-20 to + 20 mV), 350
300
ensuring sufficient electrostatic repulsion for colloidal
Size (nm)
250

stability. The higher negative charge of mixed-loaded NLs 200

150
suggests improved stability, likely due to the combined 100

effects of RLX and rutin on surface charge properties, as 50

0
indicated in Table 8. 0 24 48 72 1 week
Time
2 weeks 1 month 2 months

Free Nanoliposomes Rlx Loaded Nanoliposomes Co Loaded Nanoliposomes

Examination of nanoliposome stability at 25 °C
The stability of NLs was evaluated over two months under b
storage conditions at 25 °C, focusing on key parameters 0.9
0.8
such as PDI, particle size (nm), and zeta potential (mV). 0.7
0.6
Stability is a critical factor in determining the feasibility of 0.5
PDI

0.4
NL formulations for long-term storage and pharmaceutical 0.3
0.2
applications. 0.1
0
Size analysis revealed that free NLs exhibited values 0 24 48 72 1 week 2 weeks 1 month 2 months
Time
beyond the acceptable range after just one week and
continued to be unstable throughout the two month Free Nanoliposomes Rlx Loaded Nanoliposomes Co Loaded Nanoliposomes

(Figure 4a). In contrast, RLX-loaded NLs remained stable c

for up to two months, while co-loaded NLs stayed within 0 24 48 72 1 week 2 weeks 1 month 2 months
acceptable limits for the entire storage duration at 25 °C. 0

Similarly, PDI measurements indicated that free NLs -5

Zeta potential (mV)

became unstable after one week, whereas co-loaded NLs -10

showed instability within 72 hours. Meanwhile, RLX- -15

loaded NLs maintained stability for up to one month -20

(Figure 4b). -25

Time
Regarding zeta potential, all formulations—free
Free Nanoliposomes Rlx Loaded Nanoliposomes Co Loaded Nanoliposomes
NLs, RLX-loaded NLs, and co-loaded NLs—remained
Figure 4. Stability of the NLs over two months under storage at 25°C. a: Size
within the optimal range for NL formulations (Table 9). change, b: PDI change, and c: charge change
Throughout the storage period, zeta potential values,
which indicate surface charge and colloidal stability, were absence of additional stabilizing interactions found in
maintained within the permissible range of -20 to + 20 RLX-loaded or co-loaded systems likely contributed to
mV (Figure 4c). The specific values suggest sufficient this instability.
electrostatic repulsion to prevent significant aggregation: Statistical analysis of NL characteristics, including size,
-11.45 mV for co-loaded NLs, -3.91 mV for RLX-loaded PDI, and surface charge, revealed significant differences
NLs, and -1.14 mV for free NLs. Despite having the lowest among formulations, as confirmed by ANOVA and LSD
absolute zeta potential, free NLs exhibited instability in multiple comparisons. Particle size analysis (F = 20.75,
both size and PDI, suggesting that electrostatic stabilization P ≤ 0.001) showed that free NLs (237.07 nm, SD = 83.27)
alone was insufficient to maintain long-term stability. The were significantly larger than both co-loaded NLs

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Oleiwi et al

Table 9. Examination of nanoliposome stability at 25°C.

Standard ANOVA Multiple Comparisons LSD (P value)

Parameter NLs Mean
deviation F Sig. Test group Compared group Sig.

Co loaded NLs 144.94 10.54 Co loaded NLs RLX Loaded Nanoliposomes 0.316
Size
RLX loaded nanoliposomes 160.44 18.57 20.75 P ≤ 0.001 - Free Nanoliposomes 0.000
(nm)
Free nanoliposomes 237.07 83.27 RLX loaded nanoliposomes Free Nanoliposomes P ≤ 0.001

Co loaded NLs 0.249 0.074 Co loaded NLs RLX Loaded Nanoliposomes 0.297

PDI RLX loaded nanoliposomes 0.298 0.095 11.94 P ≤ 0.001 - Free Nanoliposomes P ≤ 0.001

Free nanoliposomes 0.467 0.234 RLX loaded nanoliposomes Free Nanoliposomes 0.001

Co loaded NLs -11.45 1.960 Co loaded NLs RLX Loaded Nanoliposomes P ≤ 0.001
Charge
RLX loaded nanoliposomes -3.910 1.400 144.7 P ≤ 0.001 - Free Nanoliposomes P ≤ 0.001
(mV)
Free nanoliposomes -1.140 2.570 RLX loaded nanoliposomes Free nanoliposomes P ≤ 0.001
PDI: Polydispersity index; RLX: Raloxifene; NLs: Nanoliposomes; LSD: Fisher's least significant difference;
F: F-Statistics

(144.94 nm, SD = 10.54) and RLX-loaded NLs (160.44 based formulations. Size analysis indicated that both free
nm, SD = 18.57). However, the difference between NLs and RLX-loaded NLs remained stable for up to two
co-loaded and RLX-loaded NLs was not statistically weeks, while co-loaded NLs exhibited stability for up to
significant (P = 0.316), suggesting that RLX incorporation one month. Despite some size increases over time due to
had minimal impact on particle size, whereas free potential aggregation or structural rearrangements, all
NLs exhibited significantly greater size variability and formulations remained within acceptable limits at 4°C
heterogeneity (P ≤ 0.001). (Figure 5a). This suggests that the combination of RLX
The last three columns show the significant pairwise and rutin may enhance structural stability by modifying
comparisons obtained using the ANOVA multiple the rigidity and composition of the lipid bilayer in the co-
comparison test. Specifically, numbers are the p-value loaded system.
for the comparison of free NLs to the appropriate Similarly, free NLs exhibited instability in PDI values
comparison group. after just two weeks of storage, whereas the PDI of
PDI analysis (F = 11.94, P ≤ 0.001) further confirmed co-loaded NLs remained stable for up to two months
that free NLs had the highest polydispersity index (0.467, (Figure 5b). In contrast, RLX-loaded NLs maintained
SD = 0.234), significantly differing from RLX-loaded NLs stable PDI values for only one week (Figure 5c, Table 10).
(0.298, SD = 0.095, P = 0.001) and co-loaded NLs (0.249, Regarding zeta potential, all formulations fell within the
SD = 0.074, P ≤ 0.001). However, no significant difference optimal range for nanoliposome stability. Notably, co-
was observed between RLX-loaded and co-loaded NLs loaded NLs displayed a higher negative charge (-12.74 mV)
(P = 0.297), indicating that both formulations maintained compared to free NLs (-7.35 mV) and RLX-loaded NLs
a relatively uniform size distribution. In contrast, free NLs (-5.14 mV) (Figure 5c). Over time, the greater absolute
exhibited the highest heterogeneity, which could lead to zeta potential of co-loaded NLs likely contributed to
instability and aggregation. enhanced electrostatic stabilization, minimizing particle
Zeta potential measurements (F = 144.7, P ≤ 0.001) aggregation and improving colloidal stability.
indicated that co-loaded NLs (-11.45 mV, SD = 1.960) had The ANOVA results (F = 1.251, P = 0.307) indicate no
the most stable surface charge, significantly differing from statistically significant difference in particle size among
RLX-loaded NLs (-3.91 mV, SD = 1.400, P ≤ 0.001) and the three formulations. RLX-Loaded NLs (229.76 nm,
free NLs (-1.14 mV, SD = 2.570, P ≤ 0.001). The lower zeta SD = 74.42), Co-Loaded NLs (165.60 nm, SD = 73.81), and
potential of RLX-loaded and free NLs suggested weaker Free NLs (199.99 nm, SD = 93.78) exhibit considerable
electrostatic repulsion, increasing the risk of aggregation. variation; however, multiple comparisons reveal that none
Additionally, the large variation in zeta potential within of the pairwise differences are statistically significant
free NLs (SD = 2.570) further confirmed their poor (P > 0.05). This suggests that the incorporation of RLX or
stability. co-loading does not significantly impact overall particle
size. The relatively high standard deviations indicate a
Examination of NL stability at 4°C broad distribution of particle sizes, which may influence
The stability of NLs was assessed under storage conditions formulation stability.
at 4°C for two months. Key parameters, including The values in the last three columns represent the
PDI, particle size (nm), and zeta potential (mV), were significant pairwise comparisons obtained from the
measured, yielding p-values of 0.307, 0.036, and 0.001, ANOVA multiple comparison test. Specifically, the 0.471
respectively. Maintaining stability at low temperatures value indicates the p-value for the comparison between the
is essential for preserving the efficacy of nanoparticle- free NLs and the respective comparison group. The value

380 Advanced Pharmaceutical Bulletin. 2025;15(2)

 Raloxifene and rutin as PEGylated Nanoliposomes

Table 10. Examination of nanoliposome stability at 4°C

Standard ANOVA Multiple Comparisons LSD (p value)

Parameter NLs Mean
deviation F Sig. Test group Compared group Sig.

RLX Loaded NLs 229.76 74.42 RLX Loaded NLs Co Loaded NLs 0.129
Size (nm) Co Loaded NLs 165.60 73.81 1.251 0.307 - Free NLs 0.471
Free NLs 199.99 93.78 Co Loaded NLs Free NLs 0.407
RLX Loaded NLs 0.400 0.150 RLX Loaded NLs Co Loaded NLs 0.018
PDI Co Loaded NLs 0.196 0.086 3.892 0.036 - Free NLs 0.728
Free NLs 0.372 0.213 Co Loaded NLs Free NLs 0.038
RLX Loaded NLs -5.140 2.440 10.98 0.001 RLX Loaded NLs Co Loaded NLs P ≤ 0.001
Zeta potential
Co Loaded NLs -12.74 4.560 - Free NLs 0.199
(mV) - -
Free NLs -7.350 2.580 Co Loaded NLs Free NLs 0.004
PDI: The polydispersity index; RLX: Raloxifene; NLs: Nanoliposomes; LSD: Fisher's least significant difference.

a The PDI measures the uniformity of particle size

450 distribution. ANOVA results (F = 3.892, P = 0.036)
400
350
indicate a statistically significant difference among
300 the formulations. Co-loaded NLs (0.196, SD = 0.086)
Size (nm)

250
200
exhibit the lowest PDI, suggesting a more uniform
150 and stable formulation. In contrast, RLX-loaded NLs
100
50
(0.400, SD = 0.150) and free NLs (0.372, SD = 0.213) have
0 significantly higher PDI values, reflecting greater size
0 24 48 72 1 week 2 weeks 1 month 2 months
Time heterogeneity. Multiple comparisons confirm significant
Rlx Loaded Nanoliposomes Co Loaded Nanoliposomes Free Nanoliposomes
differences between RLX-loaded NLs and co-loaded NLs
(P = 0.018) and between co-loaded NLs and free NLs
b (P = 0.038). However, no significant difference is observed
0.9 between RLX-loaded NLs and free NLs (P = 0.728).
0.8
These findings suggest that co-loading enhances particle
Polydispersity index (PDI)

0.7
0.6 uniformity, whereas RLX-loaded and free formulations
0.5
exhibit greater heterogeneity.
0.4
0.3 Zeta potential is a key indicator of colloidal stability.
0.2 ANOVA results (F = 10.98, P = 0.001) reveal a highly
0.1
0.0 significant difference among the formulations. Co-loaded
0 24 48 72 1 week 2 weeks 1 month 2 months
Time
NLs (-12.74 mV, SD = 4.560) exhibit the most negative
charge, indicating stronger electrostatic repulsion and
Rlx Loaded Nanoliposomes Co Loaded Nanoliposomes Free Nanoliposomes
greater colloidal stability. In contrast, RLX-Loaded
NLs (-5.140 mV, SD = 2.440) and Free NLs (-7.350 mV,
c
0 24 48 72 1 week 2 weeks 1 month 2 months SD = 2.580) display significantly lower negative charges,
0
suggesting weaker repulsive forces and a higher tendency
Zeta potential (mV)

-5
for aggregation. Multiple comparisons confirm highly
-10 significant differences between RLX-loaded NLs and
-15 Co-loaded NLs (P ≤ 0.001) and between Co-Loaded
-20 NLs and Free NLs (P = 0.004). However, no significant
-25
Time
difference is observed between RLX-loaded NLs and free
NLs (P = 0.199), indicating that both formulations exhibit
Rlx Loaded Nanoliposomes Co Loaded Nanoliposomes Free Nanoliposomes
similar colloidal stability, which is lower than that of
Figure 5. Stability of the NLs over two months under storage at 4°C. a: Size
change, b: PDI change, and c: charge change
co-loaded NLs.

1.251 refers to the F-statistic obtained from the ANOVA Lyophilization stability
test, which measures the ratio of variance between the A paired t-test was conducted to assess the impact of
groups. The value 0.307 is the corresponding p-value, lyophilization on the characterization parameters of
which indicates the level of statistical significance. Since the co-loaded NLs, including size (nm), PDI, and zeta
the p-value is greater than the typical threshold (e.g., potential (mV) (Table 11). Significant differences were
P ≤ 0.05), it suggests that the differences between the observed, with p-values of 0.023, 0.001, and 0.03 for
groups are not statistically significant. size, PDI, and zeta potential, respectively. These findings

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Oleiwi et al

indicate that the structural properties of the NLs were Morphological study
notably affected by the freeze-drying process. However, Transmission electron microscopy (TEM)
the size and charge of the NLs remained within the TEM provided valuable insights into the morphology and
optimal range after lyophilization. size distribution of the mixed NLs. TEM analysis revealed
Despite these changes, both the size and zeta potential that the mixed NLs exhibited a uniform structure with a
remained within acceptable limits for nanoparticle smooth, spherical shape and an average size of 100 ± 30.4
stability post-lyophilization. Maintaining a particle size nm (n = 15). As shown in Figure 7, RLX and rutin were
below 300 nm is crucial for enhancing drug bioavailability successfully encapsulated within the NLs. The TEM
and cellular uptake, while a zeta potential within the range images clearly demonstrated the incorporation of RLX
of -20 mV to + 20 mV provides sufficient electrostatic and rutin into the nanoliposome structure, confirming
repulsion to prevent aggregation. The ability of the NLs to their successful entrapment. The localization of these
retain these desirable properties suggests that their overall active compounds depends on their solubility properties:
colloidal stability was not significantly compromised, RLX, being relatively hydrophobic, is likely associated
supporting the lyophilized formulation’s potential for with the lipid bilayer, whereas rutin, possessing both
long-term storage and transport. However, the PDI hydrophobic and hydrophilic regions, may be distributed
exceeded 0.3, possibly due to the absence of sucrose in the between the lipid bilayer and the aqueous core. This co-
lyophilization process. loading strategy enhances the potential for synergistic
therapeutic effects and controlled drug release.
In vitro drug release assay
The release rate of RLX from NLs was significantly Cell viability assay
slower than that from the free RLX solution (Figure 6a). ER-positive BC cell line (MCF-7)
The results indicate that RLX NLs exhibited a distinctive The cytotoxic effects of RLX at various concentrations
biphasic release profile, characterized by an initial burst on MCF-7 cells were evaluated in this study using the
phase followed by a considerably slower release phase. MTT assay. The IC50 values of free RLX and its liposomal
During the first two hours, RLX molecules located on form, which inhibited 50% of MCF-7 cell viability, were
the lipid bilayer surface and not fully encapsulated within determined. Figure 8 illustrates the chemosensitivity of
the NLs contributed to the initial burst release. After four the MTT curves for (a) MCF-7, (b) MDA-MB-231, and
hours, the amount of RLX released from the free solution (c) EA. hy926 cells following 72 hours of exposure to RLX,
reached approximately 33.6% ± 1.61. However, after 24 liposomal RLX, rutin, rutin Lipo, free mix, or mixed Lipo.
hours, only 17% ± 0.97 of RLX was released from RLX Cells cultured in the medium without drug treatment
NLs, compared to around 25% ± 2.21 from mixed NLs. In (treated with a vehicle) served as controls.
comparison, 93.8% ± 1.07 of rutin was liberated in the free The IC50 value of free RLX was calculated as 9
solution within 72 hours, whereas 55% ± 1.98 was released µg/mL ± 0.191 after 72 hours. In contrast, the IC50 of
from the rutin NLs and 70.4% ± 1.20 from the mixed liposomal RLX was determined to be 40 µg/mL ± 0.13
NLs (Figure 6b). The increased release from mixed NLs
suggests that RLX-rutin interactions may influence the a
80
structural permeability of the liposomal membrane. The
70
continuous release profile observed in NLs aligns with 60
%Cumulative release

previously published findings.53,54 The sustained release 50

characteristics of NLs can be attributed to the integration 40 Rlx lipo

of RLX and rutin within the lipid bilayer, which restricts 30 RLX FREE
MIX LIPO (RLX)
20
their rapid diffusion into the dialysate. Moreover, the
10
encapsulation approach offers multiple advantages, 0
including enhanced bioavailability, reduced dosing 0 20 40 60 80
Time (hour)
frequency, and minimized dose-dependent RLX toxicity.
Consequently, encapsulating RLX and rutin in NLs may
b
serve as an effective strategy for the sustained delivery of 100
rutin in the body while simultaneously mitigating RLX’s 90
80
dose-dependent toxicity.55
% Cumulative release

70
60
Table 11. The influence of lyophilization on the characterization parameters 50 FREE RUTIN
includes size (nm) change, PDI change, and charge change 40 LIPO RUTIN
30
MIX LIPO(RUTIN)
20
Condition Particle size (nm) PDI Zeta potential (mV)
10
Before lyophilization 250 0.21 -6 0
0 20 40 60 80
After lyophilization 320 0.45 -11 Time (Hour)

PDI: Polydispersity Index. Figure 6. In vitro release assay of a: RLX and b: rutin over 70 hours

382 Advanced Pharmaceutical Bulletin. 2025;15(2)

 Raloxifene and rutin as PEGylated Nanoliposomes

Figure 7. TEM shape and size of the mixed NLs

after the same duration. These results indicate that the IC50
of RLX in the nanoliposomal (NL) form was higher than
that of the free drug. Additionally, the physical mixture
of RLX and rutin exhibited greater cytotoxicity than the
liposomal formulation. This suggests that the liposomal
preparation mitigated the cytotoxic effects of the RLX-
rutin combination.
Both the incorporation of rutin with RLX in a physical
mixture and the encapsulation of RLX within the liposomal
formulation reduced the cytotoxicity of RLX against the
MCF-7 cell line. For instance, at a concentration of 0.015
µg/mL, the survival rate of MCF-7 cells treated with free
RLX alone was 24% ± 0.016, whereas the survival rate for
the combination of RLX and rutin as a physical mixture
was 64% ± 0.05, and 70% ± 0.09 for liposomal RLX.

ER-negative BC cell line (MDA-MB-231)

Figure 8b illustrates the different concentrations of free
RLX and its liposomal form used to treat the MDA-
MB-231 cell line, along with their corresponding IC50 Figure 8. Chemosensitivity curves of (a) MCF-7, (b) MDA-MB-231, and (c)
values. After 72 hours of treatment, the concentrations of EA. hy926 cells exposed to RLX, liposomal RLX, rutin, Rutin Lipo, free mix or
mixed Lipo for 72 hours. All values are averages of triplicates + SDs
free RLX and the liposomal form were 6 µg/mL ± 0.14 and
70 µg/mL ± 0.05, respectively. The IC50 of RLX in treated to free drugs (Figure 8c). In conclusion, the addition of
cells was 31 ± 0.11 µg/mL. However, upon the addition rutin or the incorporation of RLX within the liposomal
of rutin, the IC50 increased to 36.0 ± 0.060 µg/mL in the formulation reduced cytotoxicity against both BC cell lines
physical mixture and further increased to 55.8 ± 0.008 while enhancing safety in normal human cells and tissues.
µg/mL in the liposomal mixture. The addition of rutin
did not significantly enhance cytotoxicity against the In vitro antioxidant assay
MDA-MB-231 cell line. Conversely, incorporating RLX The free radical scavenging activity of RLX, rutin, their
into liposomes reduced cytotoxicity by 1.8-fold. These mixed solution, and RLX-, rutin-, and mixed-loaded
findings indicate that the physical mixture exerted a NLs at various concentrations were evaluated. Ascorbic
greater cytotoxic effect on both BC cell lines compared to acid was used as a reference to assess radical scavenging
the liposomal formulation. capacity (Figure 9). Free radical scavenging ability is a key
indicator of antioxidant activity, which plays a crucial role
Normal endothelial cell lines in reducing oxidative stress and enhancing therapeutic
To evaluate the selectivity of the NLs, a viability assay efficacy in drug delivery systems.
was performed on normal cells (Ea. hy926) to assess The DPPH radical scavenging assay was conducted, with
their potential cytotoxic effects on non-cancerous cells. the results presented in Figure 9. The scavenging activity
The MTT assay demonstrated that the NL formulation of DPPH was maximal at 1 mg of RLX/rutin NLs, RLX/
did not induce significant cytotoxic damage compared rutin solution, RLX, and rutin. As shown in Figure 9, rutin

Advanced Pharmaceutical Bulletin. 2025;15(2) 383

Oleiwi et al

and the physical mixture exhibited enhanced antioxidant The effects of rutin on cell migration and invasion were
effects, whereas RLX alone demonstrated no antioxidant investigated. Figure 10a depicts the migration of different
activity. Notably, the mixed-loaded NLs displayed groups of MCF-7 cells after a 72-hour incubation.
antioxidant activity, indicating a potential enhancement Figure 10b shows the migration of MDA-MB-231 cells
in the combined formulation. The methanolic solution across the Matrigel surface following a 72-hour scratch
containing RLX and rutin exhibited greater antioxidant assay in various groups. The anti-migration rates of
activity than the corresponding non-combined solution, MCF-7 and MDA-MB-231 cells treated with RLX and the
suggesting a synergistic effect of co-loading both mixed-loaded NL formulations at IC50 and half-maximal
compounds. The increased antioxidant capacity of mixed- inhibitory concentrations were higher than those of the
loaded NLs highlights their potential as a multifunctional control group.
delivery system, offering both sustained drug release and The left image of the MDA-MB-231 cell line (Figure 11)
antioxidant protection. This property could be valuable represents the initial condition or an early stage following
in mitigating oxidative stress-related damage in various the wound. The right image, taken later point,
therapeutic applications. demonstrates partial wound closure. Measurements of the
wound area and perimeter indicate a progressive decrease
Migration results over time, illustrating the healing process.
Similarly, for the MCF7 cell line, the left image depicts
the wound at its initial stage, while the right image shows
partial closure at a subsequent time point. The provided
measurements of area and perimeter further highlight
these temporal changes.
MDA-MB-231 cells, known for their higher
aggressiveness and metastatic potential, exhibit different
wound-healing behavior compared to the less aggressive
MCF7 cells. Although both cell lines show some degree
of wound closure, the rate of healing may vary. The
Figure 9. Free radical scavenging activity of RLX, rutin, the mixed solution,
and RLX, rutin, and mixed NLs at different concentrations. Ascorbic acid was concentration at which a therapy inhibits 50% of cell
used as a standard for generating a radical scavenging ability calibration curve viability is referred to as the IC50 concentration. The

Figure 10. Influence of rutin on the migration and invasion of cells. Representative images of the cell migration assay. (a) Migration of MCF-7 and (b) MDA-MB-231
cells 72 hours after wounding in the different groups. The extent to which all the formulations and free drugs inhibited cell migration was calculated

384 Advanced Pharmaceutical Bulletin. 2025;15(2)

 Raloxifene and rutin as PEGylated Nanoliposomes

Figure 11. Wound closure area (2 μm) of both BC cell lines treated with mixed NL

images suggest that the NL combination affects the compare the treatment’s efficacy under controlled
wound-healing process in both cell lines, though to conditions.
varying extents. This assay is commonly used to evaluate The formulation of innovative NLs for the co-delivery
cell migration and proliferation. of pharmaceuticals is a complex process influenced by
By analyzing changes between the initial and later various factors. These parameters are crucial in defining
time points, the influence of the therapy on these cellular nanoparticle properties and drug loading efficiency,
processes can be assessed. The images indicate that at ultimately impacting the overall quality of the resulting
their respective IC50 values, the NL combination impacts formulations.56
wound healing in both MDA-MB-231 and MCF7 cell lines. The findings revealed significant differences in
Since wound closure is closely linked to cell migration and EE% and particle size between formulations using
proliferation, its modulation may reflect the therapeutic 100% chloroform as the solvent and those using a 75%
effect. To draw more definitive conclusions, additional methanol: chloroform mixture. The increase in EE%
quantitative and statistical analyses are necessary to from 51.98% to 91.29% for RLX and from 67.84% to

Advanced Pharmaceutical Bulletin. 2025;15(2) 385

Oleiwi et al

78.12% for rutin aligns with the findings of Ansari et al cytotoxic effects.
57
who investigated the effect of solvents on nanoparticle MTT cell viability assays were used to assess the cytotoxic
characterization. Additionally, the polarity of the solvents effects of RLX and rutin when administered as free drugs,
influenced encapsulation efficiency, surface charge, and physical mixtures, or NLs, using the normal endothelial
PDI, affecting the characterization of organic solvent- cell line EA. hy926. The results suggest a favorable safety
based formulations. All formulations prepared fell within profile for the nanoliposomal formulations compared
the optimal limits for size, charge, and PDI.58,59 Moreover, with free drugs. Specifically, ‘Ralox Lipo’ and ‘Rutin Lipo’
the findings demonstrated that co-loading rutin with RLX exhibited greater cell viability at increasing concentrations,
in NLs improved nanoparticle stability. These results are indicating reduced toxicity. In contrast, ‘Free Ralox’ and
consistent with previous studies,60 which also reported ‘Rutin Free’ led to significant decreases in cell viability with
enhanced nanoparticle stability through co-loading. increasing concentrations, suggesting heightened toxicity.
Co-loaded NLs carrying RLX exhibited a distinct The ‘Mix Lipo’ group also maintained greater cell viability
biphasic release profile, characterized by an initial burst at all tested concentrations, underscoring the protective
release followed by a second phase with a significantly effect of liposomal encapsulation. Ideally, for normal cell
reduced RLX release rate. This release pattern aligns lines, maintaining high cell viability even at elevated drug
with findings from previous studies employing similar concentrations is desirable, a goal achieved by liposomal
formulation methods and conditions.46,61 TEM analysis formulations. This finding highlights the enhanced safety
confirmed that the NLs were uniformly sized and of liposomal carriers, as they are designed to specifically
spherical, consistent with earlier reports using identical target cancer cells while minimizing damage to normal
preparation methods.46,60-62 cells. The liposomal formulations of RLX and rutin, both
The primary rationale for RLX therapy in ER-positive individually and in combination, demonstrate potential
breast tumors lies in its antiestrogenic effect via the ER- for safer therapeutic applications by preserving healthy
dependent pathway, initiated by the formation of the cell integrity during cancer treatment.
RLX-ER complex, which inhibits estrogen binding to the The effects of different RLX and rutin formulations on
receptor. Compelling evidence suggests that tamoxifen the migration of MCF-7 and MDA-MB-231 BC cells were
exhibits multicellular, non-ER-related actions not only in evaluated using a migration assay. The results indicate that
BC but also in other malignancies, such as hepatocellular these formulations significantly influence cell motility, an
carcinoma and lung cancer.63 important factor in metastatic potential. As expected, the
The results of this study provide strong evidence for a control groups exhibited the least migration inhibition. In
non-ER-targeted mechanism, as similar cytotoxic effects comparison, the RLX-loaded liposomal formulation (RLX
were observed in ER-positive (MCF-7), ER-negative Lipo) and the combined liposomal mixture (Lipo Mix)
(MDA-MB-231), and normal-like cell lines. Numerous demonstrated greater inhibitory effects on cell migration
studies have demonstrated that RLX influences breast, at both the IC₅₀ and 0.5 IC₅₀ concentrations, suggesting
liver, and prostate cancer cells independently of estrogen their potential in reducing cancer cell metastasis. Free RLX
receptors. RLX directly binds to the aryl hydrocarbon and the physical mixture (Mix) displayed intermediate
receptor (AhR), a molecular target that induces apoptosis effects, while the liposomal formulations showed a
in both ER-negative mouse and human hepatoma cells, pronounced improvement in migration inhibition. These
as well as in triple-negative MDA-MB-231 BC cells, findings suggest that liposomal encapsulation of RLX and
while sparing nontrans formed mammary cells.64 In rutin not only enhances solubility and safety, as previously
vivo xenograft studies indicate that RLX inhibits TNBC discussed, but also enhances their therapeutic efficacy in
growth.40 Furthermore, RLX has been shown to exert an preventing cancer cell migration, an essential factor in
alternative mechanism of action in ER-negative cell lines, controlling BC metastasis.
leading to a 27-fold decrease in EGFR expression and a Regarding the radical scavenging activity of RLX
70% reduction in Ki67 expression. This process inhibits and rutin formulations, the data indicate that free rutin
tumor cell proliferation and promotes apoptosis through exhibits superior efficacy, maintaining high inhibition
caspase-3 activation. Additionally, RLX induces apoptosis percentages across all concentrations, consistent with
in androgen-independent human prostate cancer cell its well-documented antioxidant properties. However,
lines.65 The literature supports the cytotoxicity studies liposomal encapsulation of rutin and RLX resulted in a
presented here, reinforcing the investigation of RLX as decline in scavenging activity, with a marked decrease at
a potential non-ER-targeted selective estrogen receptor higher concentrations. This effect may be attributed to the
modulator (SERM) in both ER-positive and ER-negative encapsulation altering the compounds’ interactions with
cells. This study provides compelling evidence that a free radicals. Interestingly, the liposomal mixture of RLX
nanoliposome formulation containing RLX reduces and rutin did not demonstrate the anticipated synergistic
cytotoxicity in both cell types, supporting the findings effect, instead showing a peak at an intermediate
of Oliveira et al66 who developed a novel etidocaine concentration followed by a decline. The physical mixture
formulation that enables sustained release while mitigating exhibited the least efficacy, suggesting that the free forms

386 Advanced Pharmaceutical Bulletin. 2025;15(2)

 Raloxifene and rutin as PEGylated Nanoliposomes

of RLX and rutin might interact more effectively with Supervision: Ali Al-Samydai, Aya Y. Al-Kabariti.
free radicals than their physically combined counterparts. Validation: Ali Al-Samydai, Khaldun M. Al Azzam.
Visualization: Walhan Alshaer.
These results indicate that while liposomal delivery Writing–original draft: Maryam Abdulmaged Oleiwi.
improves targeting and solubility, it may not be the optimal Writing–review & editing: Ali Al-Samydai, Aya Y. Al-Kabariti,
strategy for enhancing the antioxidant activity of RLX Khaldun M. Al Azzam, Simone Carradori.
and rutin. This underscores the importance of tailoring
formulation strategies to meet specific therapeutic Competing Interests
The authors declare that they have no conflict of interest.
objectives.
Ethical Approval
Conclusion Not applicable.
This study successfully developed and characterized
PEGylated NLs co-loaded with RLX and rutin, offering Funding
This research received no funding.
a promising drug delivery system for BC treatment.
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