Preparation and Evaluation of Co-Delivery
of Tamoxifen and Curcumin Solid Lipid
Nanoparticles (Slns) on Breast Cancer Cells MCF-7
Atefeh Naeimifar1, Saman Ahmad Nasrollahi2, Sara Dabirian3, Mahsa Shirchi4 and Ehsan Aboutaleb4*
1Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Iran
2Nanodermatology Unit, Center for Research & Training in Skin Diseases & Leprosy, Tehran University of Medical Sciences, Iran
3Department of Pharmaceutical biotechnology, School of Pharmacy, Guilan University of Medical Sciences, Iran
4Department of Pharmaceutics, School of Pharmacy, Guilan University of Medical Sciences, Iran
Submission:April 3, 2023; Published:June 14, 2023
*Corresponding author:Ehsan Aboutaleb, Department of Pharmaceutics, School of Pharmacy, Guilan University of Medical Sciences, Rasht, Iran.
How to cite this article:Atefeh N, Saman Ahmad N, Sara D, Mahsa S, Ehsan A. Preparation and Evaluation of Co-Delivery of Tamoxifen and Curcumin Solid Lipid Nanoparticles (Slns) on Breast Cancer Cells MCF-7. J Tumor Med Prev. 2023; 4(1): 555627. DOI: 10.19080/ JTMP.2023.04.555627
Breast cancer is caused by the growth of a malignant tumor mass. Luminal subtypes mainly contain estrogen and progesterone receptors. Three classes of drugs can be noted for the treatment of breast cancer, one of which is the selective estrogen receptor modulator (SERMs) such as TMX. The oral bioavailability of TMX is increased in the presence of CUR. CUR can also influence the expression of oncogenic proteins involved in the breast cancer progression. SLNs have been developed as leading nanocarriers in the treatment of cancer. SLNs containing TMX and CUR were prepared by microemulsion techniques. The average size of nanoparticles was 19.46 nm. Around 99.97% of CUR and 98.7% of TMX had loaded on nanoparticles, about 78% of TMX and 80% of CUR were released. An in vitro study of antiproliferative activity, carried out on MCF-7 cell line, demonstrated that TMX-CUR SLNs showed an antitumoral activity. The results of characterization assessments and in vitro antiproliferative activity intensely support the potential application of TMX-CUR SLNs as a carrier system at extended-release useful for breast cancer treatment. TMX-CUR SLNs had a suitable size and high loading capacity and are controlled release. They have a higher efficacy than free drugs in vitro situations.
Keywords: Breast neoplasm; Curcumin; Solid Lipid Nanoparticles; Tamoxifen, MCF-7.
Breast cancer is highly prevalent globally and is the fifth most common cancer type. It is commonly detected in women and ranks second as a cause of death amongst females. In women, breast cancer is the second principal reason of death after lung cancer. Breast cancer is more common in women between the ages of 20 and 59 In the USA, 12.5 percent of women are at risk to develop breast cancer  Meanwhile, In Asia, there is a growth in the occurrence of breast cancer due to lifestyle alterations. Mortality rates are also increasing due to the absence of screening plans and therapeutic services for breast cancer in developing countries . TMX is the first selective estrogen receptor modulator (SERM)  Oral TMX is considered as the standard adjuvant endocrine therapy for postmenopausal female with hormone receptor-positive localized breast cancer for 5 years. It decreases the risk of developing the progressive types of breast cancer in women who have had ductal carcinoma in situ. TMX binds to the estrogen receptors. It performs as an anti-estrogen agent and has lower toxicity than other chemotherapeutics . CUR is a popular natural drug, traditionally used for the treatment of a wide range of diseases derived from Curcuma longa [6,7]. Various laboratories and clinical investigations have shown the role of CUR in the prevention and treatment of several types of cancer  In studies various mechanisms of CUR as an antitumor agent such as apoptosis  and cell cycle arrest  have been approved.
Hence, co-administration of TMX with CUR can lead to
improved/superior treatment programs. The combination of
active ingredients that sensitize cancer cells to chemotherapeutics
has been well known as an efficient method of improving the
efficiency of the drugs  Natural compounds from medicinal
plants are mainly safe and low toxicity . For the last several
decades, the pharmaceutical industry has had various problems
in developing poorly soluble active ingredients into dosage forms.
Numerous approaches including solid dispersion in carriers,
particle size reduction, salt formation and prodrug, polymorphism,
complexation, and solubilization have been used to increase the
solubility of poorly water-soluble active ingredients  TMX
and CUR are cases of such molecules that are highly permeable
but poorly soluble. Among these methods, for making better
solubility, preparation solid dispersions over hot melt mixing,
solvent methods, and melt extrusion has achieved acceptance.
SLNs were first introduced by Muller. They are a colloidal system,
composed of biological lipid and emulsifiers that are solid at
room condition . SLNs are a substitute vehicle system for
liposomes and polymer nanoparticles. Another benefit of SLNs
is that they improve biodegradability of drugs. They consist of
biocompatible and physiological lipids, which are suitable for
the incorporation of hydrophilic, lipophilic, and poorly watersoluble
active ingredients. Their ability to decrease the particle
size offers promising prospects in making them vehicles for
effective, sustained release, and extended stability of the drug
. The main aim in this study was to improve the dissolution
and bioavailability of TMX when used in combination with CUR in
the treatment of breast cancer on the MCF-7 cell lines.
TMX and Sodium bicarbonate were purchased from Sigma
Aldrich (Canada). CUR and Polysorbate 80 (tween 80) were
bought from Merck (Germany). Cetyl palmitate was obtained from
Oleon (Italy). DMEM high glucose medium, Gentamycin, and FBS
were provided from Gibco (Maryland, United States). MCF-7 cells
were attained from the Pasteur Institute (Tehran, Iran).
Fabrication of TMX-CUR SLNs
TMX-CUR SLNs were prepared using the microemulsion
cooling method. In brief, to prepare TMX-CUR SLNs formulation,
lipid phase (cetyl palmitate) was heated to 65℃, then tween 80
as a surfactant was added and after the mixing was completed,
TMX powder was added to lipid phase and stirring continued to
dissolve TMX entirely. Afterwards, the aqueous phase (containing
CUR, tween 80, and distilled water) was heated up to 65℃ and was
added to the lipid phase and was mixed at 1400 rpm. Following
fabrication of Nano emulsion, the mixture was sonicated using
a probe ultrasonic (Hielscher up 400s, Germany) with 0.6 cycle
and 200-watt power for 3 minutes. Ultimately, the prepared
nano emulsion was diluted gradually by deionized water on the
magnetic stirrer at 4-8℃ in the ratio of 1:1 to fabricate the TMXCUR SLNs.
The TMX-CUR SLNs were well-diluted in distilled water with
the ratio of 1:10 and analyzed for average Zp, particle size, and
polydispersity index (PdI) using zetasizer (Nano flex, Particle
meter, UK). For analyzing Zp, at first optical refractive index was
measured by Abbe refractometer (AR2008, A.Kruss optronic,
Germany). All the results resemble the average ± SD of three
different tests at 25±2℃.
Transmission Electron Microscopy (TEM)
TMX-CUR SLNs was specified in terms of their size, structure,
and surface morphology by TEM, utilizing an electron microscope
(Zeiss EM10C, Germany). Nanoparticles were coated by a thin
film of uranyl acetate 2% and then electron photography was
performed by the electron microscope.
Powder X-Ray Diffraction (P-XRD)
P-XRD measurment was performed to prove the formation
of a new solid-state. P-XRD analysis was conducted for SLNs
without the drug (placebo) and TMX-CUR SLNs, using an X-ray
diffractometer X’Pert PRO MPD (PANalytical, Holland). Cu K𝛼
radiation in the scanning range of 2𝜃 = 5–80՞ was used in room temperature and pressure.
Entrapment efficiency (EE)
EE was analyzed as the ratio of the trapped drug in the carrier
system over the total drug used in the preparation of SLN. LC
was explicated as a ratio of drug-loaded in carriers to the lipid
of carriers. To calculate EE with a spectrophotometer, the first
calibration curve of different concentrations of each drug in solvent
(deionized water and tween 80) was carried out. Then, 10 ml of
TMX-CUR SLNs suspension was precisely weighed. The sample
was centrifuged at 4000 rpm for 10 min by centrifuge (TDZ5-WS,
Selecta Lab). The absorbance of the supernatant was measured
using the Fluorescence spectrophotometer instrument (cary
eclipse, Agilent) and UV-VIS spectrophotometer instrument (Cary
60, Agilent) at 360 nm and 361 nm for TMX and CUR, respectively.
Finally, the mass of each active ingredient was analyzed with
the standard calibration curve. The EE and drug loading of the
fabrication were measured by the following equations for each
where “M initial drug” is the mass of the initial drug, “M free
drug” is the mass of the free drug present in the supernatant.
“WDL” is the weight of drug-loaded in nanoparticles and “WNP” is
the weight of nanoparticles (lipids).
The in vitro drug release studies were carried out using the
dialysis bag technique. 5 ml of TMX-CUR SLNs suspension was
put into the dialysis bag (10 KD cut-off, Merck) which was then
placed inside a screw cap glass beaker filled with release media
(deionized water, tween 80 at the ratio of 33:1) with pH =7,
agitated at 100 rpm and 37 °C in a shaking water bath. 3 ml of
samples were withdrawn and analyzed spectrophotometrically.
The percentages of the drug release from nanoparticles were
plotted against time intervals 0, 0.5, 1, 2, 4, 8, 24, and 48 h for each
drug separately. The following formula was used to measure the
drug release percentage:
Breast cancer cell line MCF-7 was obtained from the National
Center for Genetic and Biological Reserves in Iran and cultured
in DMEM medium supplemented with 10% FBS. The cells were
maintained in an incubator in 37 °C, 5% CO2 concentration and
95% relative humidity (Memmert GmbH, Germany). The medium
was altered every 2-3 days. The cell lines were subcultured when
they were around 80–90% confluent with 0.25% trypsin. The cells
were classified into eight groups as follows:
• Free TMX: treated with 40 μmol/mL
• Free CUR: treated with 40 μmol/mL
• Free TMX-CUR: treated with 40-40 μmol/mL
• TMX SLN: treated with 40 μmol/mL
• CUR SLN: treated with 40 μmol/mL
• TMX-CUR SLN: treated with 40-40 μmol/mL
• SLN: treated with unloaded SLN
• Control: without any treatment
MTT assay was used to compare the Cytotoxic effect of study
groups on cell viability. In brief, MCF-7 cells (1 × 104 cells/well)
were cultured in 96-well plates. After treatment and further
incubation for 72 hours, the MTT solution at a concentration of
0.5 mg/mL was added to each well and kept at 37 °C for 3 h. After
removing the supernatants, 150 μL of DMSO was added to each
well. Using a microplate reader (Epoch) absorbance at 570 nm
was determined. To measure the toxic effect of groups on the MCF-
7 cells, IC50 values were analyzed by MTT assay. The IC50 value
of free TMX was calculated using GraphPad Prism (version 5, USA)
software with a dose-response curve after 72 hours. The viable
cells percentage was calculated using the below equation:
Percentage of viable cells= (ODTest / ODcontrol) × 100
The data achieved was subjected to statistical analysis. The
alterations in mean values among the groups were expressed as
mean ± standard deviation. Data analysis was examined by using
Paired sample t-test and equivalency and noninferiority test. The
entirety of statistical calculations is taken using Statgraphics18
(version 12, USA) software. Moreover, a P-value of ≤0.05 is
considered statistically significant.
Developments in drug delivery systems including
nanoparticle-based vesicular drug delivery systems have
improved various factors such as solubility, bioavailability,
stability, release properties, and improve the therapeutic effects
of already-efficient molecules. Principally, SLNs have developed
as promising nanocarriers in cancer therapy. SLNs accelerate the
cellular uptake of the incorporated drugs through the modulation
of active, passive, and cotransport mechanisms . Drugs are not
specific against tumor cells; therefore, they accumulate not only in
tumor cells but also in healthy tissues. New approaches that can
deliver active ingredients more specifically and make less toxicity
are required .
The use of TMX-CUR SLNs to improve the physical,
pharmacokinetic characteristics and antitumor efficacy is one such
approach. TMX and CUR are efficient and multitarget ingredients
for the prevention and treatment of numerous cancers, such as
breast cancer . In this study, the microemulsion technique
was chosen because of its accessibility and appropriate particle
characteristics. TMX and CUR were successfully incorporated into
SLNs. The average size of TMX-CUR SLNs was (found at REMOVE)
19.46±1.45 nm with a PdI of 2.70±0.04 nm. The surfaces of TMXCUR
SLNs carried a negative charge due to the presence of cetyl
palmitate. The Zp was -21.9±0.07 mV. Particle size is a major
characteristic property to be examined in a nanometric system
to ensure the efficiency of drug delivery. Abbasalipour Kabir et
al.  fabricated SLN of TMX and reported that the average size
of preparation was around 251.6 nm . Senthil Kumar et al
prepared SLN of CUR with the particle size, polydispersity index,
zeta potential of 214.60± 3.55nm, 0.49±0.03, -29.63±0.50 mV,
respectively . Our results were concordant with the findings
of this paper.
The PdI measures the width of size distribution and
homogeneity of droplet size, which is usually about 0.5 or less
. The Zp was stated as a function of surface properties of
particles and can be used to forecast their long-term stability .
An upper value of Zp has shown the stability of the dispersion 
Righeschi et al.  and Marangoni et al. showed that Zp of CUR
nanoparticles were -33 mV and -29.6 mV, respectively [24,25].
Furthermore, Al Haj et al. revealed Zp of TMX nanoparticles of
-15.4 mV ..
TEM results revealed that the TMX-CUR SLN had spherical
shapes with smooth round edges. The results also demonstrated
their nanoscale size range [Figure 1]. The EE depends on the
physicochemical properties of formulation, the reaction between
drug and vehicle, and drug and membrane. TMX-CUR SLN
showed EE of 97.48% and 99.41% for TMX and CUR, respectively.
Moreover, TMX-CUR SLN showed DL of 9.77% and 9.76% for
TMX and CUR, respectively. Our results established that the
almost complete EE showed high efficiency of SLN in taking up
the drugs. The considerably higher EE might be recognized for its
lipophilic nature and is indicative of the formulation and method
As P-XRD results shown TMX, CUR powder, and cetyl
palmitate showed a series of sharp peaks verifying its crystalline
nature. However, in TMX-CUR SLN and placebo SLN, these peaks
disappeared, indicating the amorphous nature of the particles
[Figure 2]. Comparison of area under the P-XRD peaks of TMX-CUR
SLN and placebo SLN showed the area under the peak of TMX-CUR
SLN is larger than placebo SLN which demonstrates high EE of
TMX-CUR SLN and changing crystalline phase.
Drug release kinetics involve the application of mathematical
models to predict the drug release profile. The correlation
coefficient (R2) of various kinetic models was calculated for
TMX-CUR SLNs. First-order and Weibull kinetic models obtained
the best model with the linearity of R2=0.936 for CUR-SLN and
R2≥0.9853 for TMX SLN, respectively. First-order kinetics refers
to drug release that is concentration-dependent and the Weibull
model the logarithm of the released drug and logarithm of the time
would have a linear relationship. The Weibull equation mostly fits
all kind of dissolution curves and builds up the fraction of the drug
(m) when applied the equation to release of drugs . As shown
in the results, after 48 hours of in vitro releasing 78% of TMX and
80% of CUR were released [Figure 3]. These two release patterns
showed the slow release of TMX and CUR from preparation.
Furthermore, the release of CUR was slower in the beginning
compared to TMX. These results confirmed that the extended
and completed release pattern of TMX-CUR SLNs fabrication was
attained. SLNs were dispersed in a liquid lipid medium which was
enclosed by a solid lipid shell. This structure consequently offered
the extended-release profile of the drug from nanostructure. This
controlled release pattern can decrease the adverse reaction of
drugs. In another in vitro release study, fabricated TMX loaded
SLNs demonstrated a similar release . Cell survival and
apoptosis are regularly applied to assess the efficacy of anti-cancer
drugs. Anticancer active ingredients commonly kill dividing cells
by stimulation of the apoptosis process . In this study, we
have revealed that TMX-CUR SLN is effective in decreasing the
population/number of MCF-7 cells due to growth suppression and
stimulating cell death. After treatment cells by 40 μm of TMX SLN,
CUR SLN, Free TMX-CUR, and TMX-CUR SLN the percentage of cell
viability were 53.12 ± 1.55, 39.18 ± 2.04, 52.50 ± 0.49, and 42.54 ±
8.95, respectively [Figure 4].
The IC50 of TMX-CUR SLNs was normally lower than that
of free TMX-CUR. In the presence of SLNs, the viability of cells
reduces, and the cancer cells lose their normal morphological
characteristics, detach, aggregates, and later develop apoptotic
bodies [Figure 5].
The results showed that TMX-CUR SLNs have an equally
efficient cytotoxic activity against MCF-7, compared to CUR
SLNs. CUR-SLNs had the maximum toxicity effect on the cell line.
Furthermore, the results showed that drug-loaded SLNs have
more effective cytotoxic activity against MCF-7 than the free drug.
Ho WY et al.  and Chowdhuty et al.  in two different studies
showed that IC50 of TMX after 72 hr were 27.46 μM and after 48
hr was 30.11 μM, respectively [30,31]. The improved preventive
effect of TMX-CUR SLNs on the growth of MCF-7 In comparison
with Free TMX-CUR group cells may be related to the lipophilic
characteristic of the carrier, which stimulates the intracellular
uptake . Fontana et al.  showed characterization
assessments and antiproliferative activity intensely support
the possible application of tamoxifen-loaded SLNs as a carrier
system at sustaned-release useful for treatment of breast cancer.
Furthermore, other studies revealed that TMX loaded SLNs have a
better effect than free drug [33-36]. The major limitation of this
research was hardly providing chemical materials. Lack of short
time and long-term stability study is another limitation that would
possibly affect the reliability of the data.
In this study, we proposed a new pharmaceutical formulation
of SLN for TMX and CUR in combination with appropriate
characteristics results. This study was conducted to attain
larger amounts of TXM and CUR loaded into SLNs with extended
drug release profile. The cytotoxicity study revealed that the
formulation was as toxic as free drugs on MCF-7 cell lines.
The procedure performed in this case report was in accordance
with the ethical standards of the institutional and/or national
research committee and with the 1964 Helsinki declaration and
its later amendments or comparable ethical standards.
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Ho WY, Yeap SK, Ho CL, Raha AR, Suraini AA, et al. (2011) Elephantopus scaber induces cytotoxicity in MCF-7 human breast cancer cells via p53-induced apoptosis. Journal of Medicinal Plants Research 5(24): 5741-5749.