Research Paper

Synergistic Therapeutic Strategy of Dual Drug-loaded

Lipid Polymer Hybrid Nanoparticles for Breast Cancer
Treatment
T. H. TRAN1, H. T. NGUYEN2, C. S. YONG2, D. H. TRUONG3, J. O. KIM2*
Department for Management of Science and Technology Development, 1Faculty of Pharmacy, Ton Duc Thang University, Ho
Chi Minh City, Vietnam, 2College of Pharmacy, Yeungnam University, 214-1, Dae-Dong, Gyeongsan 712-749, South Korea,
3
Institute of Research and Development, Duy Tan University, 03 Quang Trung, Da Nang, Vietnam

Tran et al.: Dual Drug-loaded Lipid Polymer Hybrid Nanoparticles

In this investigation, a smart nanocarrier-loaded docetaxel, a microtubules disrupting agent and vorinostat,
a histone deacetylase inhibitor was developed to achieve a synergistic anticancer effect. Dual drug-loaded
lipid polymer hybrid nanoparticles were prepared, with easy fabrication and favourable properties including
small size, narrow distribution and a high loading efficacy. The in vitro drug release conducted in phosphate-
buffered saline, pH 7.4 and acetate-buffered saline, pH 5.5 media demonstrated the sustained, pH-dependent
release profile. The nanoparticles were effectively taken up by cells, which ensured greater suppression of cell
growth. The co-delivery of both drugs exhibited a synergistic effect on the induction of cancer cell apoptosis,
resulting in greater inhibition of SCC-7, MCF-7, and MDA-MB-231 cancer cells by the drug-loaded carrier.
These promising results may lead to clinical applications with enhanced docetaxel activity.

Key words: Docetaxel, vorinostat, combination therapy, lipid carrier, pH sensitive polymer

With an estimated 1.67 million new cases diagnosed DTX treatments. The alteration of gene expression
in 2012, breast cancer is considered to be the second through DNA methylation and histone modification
most common cancer type in the world. In terms of is considered to be a cause of resistance to DTX in
abnormal cell growth, it presents the most severe threat cancer[12] where histone deacetylase inhibitors fill
to women[1,2]. One treatment strategy is chemotherapy the gap for improving cancer treatment. In addition,
with docetaxel (DTX, Taxotere), the most common treatment with VRS has also been shown to increase
anticancer drug used against breast cancer[3,4]. DTX has the cytotoxicity of various therapeutic agents, including
been reported to induce cell cycle arrest and apoptosis Taxol, through the relaxation of the chromatin structure
through binding and inhibition of the depolymerisation and an increase in the sensitivity of a drug to its DNA
of the α-tubulin subunit of microtubules[5]. Although it target[13].
has contributed greatly to antibreast cancer therapies,
Despite the enhancement of therapeutic efficacy,
resistance to DTX has become a risk to patients[6,7].
combinational strategies are challenged by a variety
Among the many strategies to overcome this drawback,
of different drug pharmacokinetics, which may end
combinatorial formulations, with the co-delivery
up with an inconsistent drug uptake and suboptimal
of two drugs, have been successfully developed to
drug combination at the tumor sites. In the last
reverse multiple drug resistance in in vitro as well as
decade, nanoparticles drew a great attention not only
in vivo cancer models[8-10]. In case of DTX, Shi et al.
for efficacy improvement but also for restriction of
investigated a combination with vorinostat (VRS),
a histone deacetylase inhibitor, to enhance cell death
This is an open access article distributed under the terms of the Creative
in breast cancer[11]. In detail, VRS enhanced the Commons Attribution-NonCommercial-ShareAlike 3.0 License, which
DTX-induced G2/M arrest in breast cancer MDA- allows others to remix, tweak, and build upon the work non-commercially,
as long as the author is credited and the new creations are licensed under
MB-231 cells whereas the expression of cell cycle and the identical terms
apoptosis-associated proteins in MDA-MB-231 cells
Accepted 02 April 2019
are noticed to be changed in association with VRS/
Revised 10 December 2018
Received 21 August 2018
*Address for correspondence
E-mail: [email protected] Indian J Pharm Sci 2019;81(3):474-482

May-June 2019 Indian Journal of Pharmaceutical Sciences 474

 www.ijpsonline.com
drawback such as experimental multidrug resistant bovine serum and 1 % penicillin-streptomycin (Hyclone
or administration variety[9]. Methoxy-poly(ethylene Laboratories, Logan, UT, USA), respectively. Cells
glycol)-block-poly(L-aspartic acid) (PEG-b-PAsp) were maintained at 37° in an atmosphere of 5 % CO2.
is a linear amphiphilic block co-polymer able to The squamous cell carcinoma (SCC-7), human breast
self-assemble into nanoparticles with a hydrophobic adenocarcinoma (MCF-7), and human Caucasian
poly(L-aspartic acid) core and a hydrophilic PEG breast adenocarcinoma (MDA-MB-231) cells were
shell[14,15]. Owing to the protonation of the carboxylic originally obtained from the Korean Cell Line Bank
group at low pH, anionic poly(L-aspartic acid) has been (Seoul, South Korea).
studied as potential pH-sensitive drug carrier[16,17]. The
combination of this polyelectrolyte block copolymer Preparation of lipid polymer hybrid nanoparticles:
on the surface of nanoparticles could be potential to The modified emulsification method was used
boost the drug release in the acidic environment found to fabricate lipid polymer hybrid nanoparticles
in the endosomes of cancer cells[15,16,18-20]. Furthermore, (LPHs)[23]. Briefly, VRS and DTX were dissolved in
the PEG shell could enable prolonged blood circulation melted lipid mixture consisted of Capryol 90, TPGS,
and avoid the rapid elimination of nanoparticles[21,22]. DDAB in the ratio of 3:1:0.05. Then lipid solution was
In this work, both drugs were incorporated into the lipid homogenously dispensed into 2 mg/ml PEG-b-PAsp
core of a smart carrier, which was designed to revert from solution using Ultra Turrax® T-25 homogenizer (IKA®-
a favourable negative charge into a positive charge in Werke, Staufen, Germany) for 3 min at 13 500 rpm.
acidic environment to improve binding of nanoparticles The sonication process at 90 % amplitude was applied
to the surface of cancer cells. The dynamic properties to above dispersion using a high-intensity probe
of carriers including particle size and morphology were sonicator (Vibracell VCX130; Sonics, Newtown, CT,
characterized using dynamic light scattering (DLS) and USA) for 5 min. The final suspension was purified
transmission electron microscopy (TEM), respectively. by using a centrifugal tube (molecular weight cut-off
In vitro drug release was evaluated to assess the 30 kDa) after cooling on ice.
dissolution rate of both drugs into different conditions.
Lyophilization of LPHs:
In addition, intracellular uptake was analysed using
confocal imaging and flow cytometry and the in vitro The LPHs dispersion was lyophilized using mannitol
cytotoxicity was conducted to check the activities of as a cryoprotectant (FDA5518, IlShin, South Korea).
the drugs in single or combination. Finally, apoptosis Before lyophilization, the dispersion was frozen at
studies were conducted to provide stronger evidence –20° for 3 h before further freezing at –80° overnight.
using nanoparticle-based combination therapy for the Lyophilization was conducted at a temperature of −25°
treatment of breast cancer. for 24 h, followed by a secondary drying phase for
12 h at 20°.
MATERIALS AND METHODS
Physical characterization of nanoparticles:
DTX and VRS were purchased from LC Laboratories
A Zetasizer Nano-ZS90 was used to measure the
(MA, USA). Capryol 90 (Capryol) was from
Gattefosse (Cedex, France). Coumarin-6 and Hoechst particle size and zeta-potential of the nanoparticles.
33342 were purchased from Thermo Fisher Scientific The formulation was diluted by distilled water prior
(Waltham, MA, USA). 3-(4,5-Dimethylthiazol-2- to three replicate measurements. After negative
yl)-2,5-diphenyl-tetrazolium bromide (MTT) and staining dispersion with 2 % (w/v) phosphotungstic
didecyldimethylammonium bromide (DDAB) were acid, nanoparticles were placed into copper grid and
obtained from Sigma (St. Louis, MO, USA). D-α- observed by TEM (H7600, Hitachi, Tokyo, Japan)[24].
tocopherol polyethylene glycol 1000 succinate (TPGS) To determine the quantity of drugs encapsulated in
was supplied by Isocheim (Vert le Petit, France). PEG- LPHs, an Amicon centrifugal ultrafiltration device
b-PAsp (MW: 12 000) was purchased from Alamanda (MWCO 10 kDa, Millipore, Billerica, MA, USA) was
Polymers (Huntsville, AL, USA). All other chemicals used to separate dispersion. The centrifugation was
were of reagent grade and were used without further introduced for 15 min at 5000 rpm. The free drug in
purification. the bottom chamber of the device was collected and
All cell lines were cultured in Roswell Park Memorial evaluated by using HPLC (Hitachi Ltd., Tokyo, Japan)
Institute-1640 medium supplemented with 10 % fetal with a UV/Vis detector (model L-2420). Each drug was
475 Indian Journal of Pharmaceutical Sciences May-June 2019
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analysed individually, DTX was eluted using a mobile period of 3 h. The crystals in each well was dissolved in
phase (acetonitrile and phosphate buffer (pH=3) at a 100 μl DMSO and the absorbance was measured using
volume ratio of 60/40) and detected at wavelength of a microplate reader at 570 nm (Multiskan EX, Thermo
232 nm[25] and for VRS, the mobile phase consisted Scientific, Waltham, MA, USA). Cell viability was
of formic acid (0.1 %)/acetonitrile (60/40) and the calculated from the following formula, cell viability
detection wavelength was 241 nm[26]. (%) = [OD570(sample)–OD570(blank)]/[OD570(control)–
OD570(blank)]×100.
To depict the crystal pattern of samples, the
CuKα radiation source of X-ray diffractometer (X’Pert CalcuSyn software (Biosoft, Cambridge, UK) was used
PRO MPD; PANalytical, Almelo, the Netherlands) to calculate combination index based on the median-
was used to generate X-ray laser with a voltage of effect principle for each combination corresponding
40 kV, a current of 30 mA, and a scan step size of to the cell viability from the MTT assay. Additivity,
0.02. The measurement was conducted three times; a synergy, and antagonism were assigned according to
representative pattern is shown[27]. CI values of 1.0, <1.0, and >1.0, respectively[32,33].
The in vitro drug release study was conducted in two Induction of apoptosis in cells treated with drugs was
different pH conditions (phosphate-buffered saline, determined using the Annexin-FITC/PI kit, which
pH 7.4 and acetate-buffered saline, pH 5.5) under sink classifies the cells into four quadrants; subsequently,
conditions at 37°. Two millilitres of formulation were the percentages of early apoptotic cells (FITC positive/
added into dialysis membrane tubing (Spectra/Por; PI negative), late apoptosis (FITC/PI positive), and
3.5 kDa cutoff, CA, USA) and placed in 35 ml of release necrotic (FITC negative/PI positive) were calculated.
medium. At predetermined intervals, 0.5 ml samples of For sample preparation, a 6-well plate containing 1×105
the release medium were withdrawn and another 0.5 ml cells/well was incubated for 24 h. Afterwards, each well
fresh medium was subsequently added to maintain the was treated with the individual formulations, including
volume. The drug in the release medium was quantified free VRS, free DTX, free VRS/DTX combination, and
by the HPLC method as described above[14,28]. VRS/DTX-LPH for 24 h at a concentration of 20 μM.
The treated cells were harvested and rinsed before
In vitro anticancer activity of nanoparticles: dispersing in 100 μl binding buffer and stained with PI
Flow cytometry was used to examine the cellular (10 μl, 1 mg/ml) and Annexin-FITC (5 μl), vortexed
uptake of LPHs. Coumarin-6, a fluorescent dye, was and kept for binding. After 30 min, an additional
incorporated into LPHs to observe the internalization 200 μl binding buffer was added and the suspension
in SCC-7, MDA-MB-231 and MCF-7 cell lines. For was analysed on a FACS-Verse flow cytometer (BD
this, cells were seeded in 12-well plates at the rate Biosciences, San Jose, CA, USA)[34,35].
of 10×104 cells per well and incubated overnight.
The process of apoptosis was investigated by staining
Subsequently, the cells were treated with Cou6-LPHs
the cells with Hoechst 33342 (Sigma, USA). Cells
at different concentrations and incubation times. The
were seeded at the rate of 105 cells/well in a 12-well
cells were then trypsinised, collected, and suspended
plate. After incubation for 24 h, each well was treated
in flow cytometry buffer. The fluorescent intensity of
with various samples, including free VRS, free DTX,
the particles taken up was detected by using a FACS-
free VRS/DTX combination, and VRS/DTX-LPH at
Verse flow cytometer (BD Biosciences, San Jose, CA,
2 μM. Afterwards, the cells were rinsed twice with
USA)[29,30]. PBS and fixed in 10 % formaldehyde PBS for 20 min
The MTT assay was utilized to examine the toxicity at room temperature. Cells were washed again before
of single drugs, combination drug, and formulations staining with Hoechst 33342 for 15 min in the dark.
in cancer cell lines[7,31]. Cells were seeded in each The Hoechst 33342-stained nuclei were observed on a
well (5×103 cells/well) of a 96-well plates and grown fluorescence microscope (Nikon Eclipse Ti)[36,37].
overnight. The cells were then treated with free VRS,
RESULTS AND DISCUSSION
free DTX, the combination of free VRS and DTX,
and VRS/DTX-LPH for 24 h. Drug-containing media As VRS and DTX are both hydrophobic drugs, they can
were aspirated carefully and replaced with fresh be carried on a lipophilic, biocompatible vehicle with
growth media containing MTT solution (1.25 mg/ml); a high loading capacity to amplify their activity[4,38,39].
subsequently, the cells were incubated for a further In the past few decades, lipid carriers have proven
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an attractive option through the incorporation of cell membrane to increase uptake into the endosome[14].
hydrophobic agents into the lipophilic matrix as The morphology of VRS/DTX-LPH was visible in the
an effective delivery system into the body[40,41]. In polymeric shell and the lipophilic core was seen in the
this study, Capryol 90, TPGS, DDAB was used to TEM image (fig. 1B). Moreover, the estimated particle
dissolve VRS, as well as DTX, with a high entrapment size and size distribution observed in morphological
efficiency (73.7±2.1 % and 75.8±6.8 %, respectively). images were consistent with DLS results.
The total content of the two drugs was 3.46 % at a
The XRD data of free VRS, free DTX, blank LPH, and
VRS/DTX molar ratio of 2:1. The ratio between the
two drugs was selected owing to their biological VRS/DTX-LPH are shown in fig. 1C. Numerous sharp
synergism. In addition, the fabricated nanoparticles and intense peaks at various 2θ scattered angles indicated
demonstrated favourable properties, such as small the highly crystalline nature of both drugs, whereas
size (232.4±5.8 nm; fig. 1A), narrow distribution those characteristic peaks disappeared in the case of
(0.297±0.09), and negative charge (–21.8±2.9 mV). VRS/DTX-LPH. This absence could be explained
The negative charge of formulated particles indicated by the transformation of drugs to an amorphous and
the presence of PEG-b-PAsp on the surface of the molecular dispersion[44]. The transformed state of drug
nanoparticles. The PEG chain prolonged the residence indicated the enhancement of drug distribution inside
of carriers in the blood stream by avoiding opsonisation the lipid core via hydrophobic interaction, which could
from the macrophage system[42,43]. In addition, in the increase drug solubility and contribute to preservation
mimic acidic environment of tumours, PEG-b-PAsp of drugs for later release at the target site.
undergoes protonation-deprotonation cycles and its pH-dependent release profile of the dual drug-
protonation destabilizes the polymeric shell of LPH[14]. incorporated LPH (VRS/DTX-LPH) is presented in
Consequently, VRS/DTX-LPH exposed the cationic fig. 1D. Under equivalent conditions, the release rate of
core, which could interact with the negatively charged VRS was significantly faster than that of DTX (p <0.01).

(A) 18 (B)
16
14
Intensity (Percent)

12
10

8
6

4
2

0
100 1000 200 nm
Size (d.nm)

(C) (D)60
50
Drug release (%)

40
Intensity (cps)

30
**

20

10

0
0 10 20 30 40
Time (h)
10 15 20 25 30 35 40 45 50
2 Theta (°)

Fig. 1: Physical properties of VRS/DTX-LPH nanoparticles

(A) Particles size and distribution, (B) Morphological images, (C) X-ray diffraction patterns, (▬▬) VRS; (▬▬) DTX; (▬▬) blank
LPH; (▬▬) VRS/DTX-LPH, and (D) in vitro drug release in ABS pH 5.5 and PBS pH 7.4, (▬▲▬) VRS (pH 5.5); (▬●▬) VRS (pH
7.4); (▬♦▬) DTX (pH 5.5); (▬■▬) DTX (pH 7.4). Data expressed as the mean±SD (n=3); **p-value <0.01
477 Indian Journal of Pharmaceutical Sciences May-June 2019
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This could be attributable to the higher hydrophobicity intracellular uptake using flow cytometry techniques.
of DTX (logP ~3) than VRS (logP ~2)[45,46]. In addition, When the coumarin-6 concentration was increased from
there was a remarked increase in the percent VRS and 1 to 5 μg/ml, the fluorescence intensity respectively
DTX released from LPH in acidic conditions (pH 5.5) increased in all cell lines, which demonstrated the
compared to that released under normal physiological increase of penetrated particles into cancer cells
environment (pH 7.0) after 24 h (p<0.01 in comparison (fig. 2A). In addition, fig. 2B illustrated the accumulation
of release percent in acidic and neutral pH of DTX). This of carriers in the cells after 15 min, which indicated
phenomenon could be modulated by the protonation the rapid internalization that increased with an increase
of the carboxylic group on the poly(L-aspartic acid) in incubation time (30 and 60 min). Compared to the
chains on the LPH surface[14,16]. Over 36 h, VRS and untreated cells, a remarkable increase in fluorescence
DTX were slowly released, approximately 42 and intensity was recorded in all cell lines. Collectively, the
25 % release occurred at pH 5.5, compared to 30 and data suggested that the uptake of carriers followed a
15 % at pH 7.4, respectively. The slower release time- and dose-dependent pattern.
in normal conditions may minimize the toxicity of The MTT assay was conducted to evaluate the effect of
anticancer drugs; conversely, once the drug was the individual drugs, drug combination, or drugs loaded
internalized into the endosome, additional release was into LPH on different cell lines. Most treatments resulted
triggered by the acidic environment. This strategy is in a dose-dependent effect in which the cell viability
considered as passively targeted drug delivery, enabling was reduced when the concentration increased (fig. 3).
safer and more effective therapy. Although the dose-dependent pattern was common to
The intracellular uptake of LPHs was characterized both drugs, the effect was not significantly different in
in vitro in three different cell lines (squamous cell MCF-7 and MDA-MB-231 when the cells were treated
carcinoma, SCC-7; human breast adenocarcinoma, by DTX. This phenomenon could be explained by the
MCF-7; human Caucasian breast adenocarcinoma, resistance of breast cancer cells to Taxol through the
MDA-MB-231). Firstly, coumarin-6-loaded escape of apoptosis pathways, efflux of the drug, or
nanoparticles were incubated with cells to observe alteration of drug-binding sites[6,7]. Among the three

(A) SCC-7 MCF-7 MDA-MB-231

Normalized To Mode
Normalized To Mode

Normalized To Mode

0 0 0
0 1 2 3 4 0 1 2 3 4 0 1 2 3 4
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

FL1-H FL1-H FL1-H

(B) SCC-7 MCF-7 MDA-MB-231

Normalized To Mode
Normalized To Mode

Normalized To Mode

0 0 0
0 1 2 3 4 0 1 2 3 4 0 1 2 3 4
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

FL1-H FL1-H FL1-H

Fig. 2: Uptake of Cou-6-LPH nanoparticles as assessed by flow cytometry in SCC-7, MCF-7, and MDA-MB-231
Coumarin-6 uptake occurred (A) in a dose-dependent manner, ( ) control; ( ) 1 μg/ml; ( ) 2 μg/ml; ( ) 5 μg/ml and (B) in a
time-dependent manner, ( ) control; ( ) 15 min; ( ) 30 min; ( ) 60 min
May-June 2019 Indian Journal of Pharmaceutical Sciences 478
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cell lines, MDA-MB-231 was the least sensitivity treatments, the combination of VRS and DTX induced
to both VRS and DTX. In detail, at concentration higher cytotoxicity, especially in MDA-MB-231 cells.
of 30 μM, the cell viability of MDA-MB-231 was In contrast, in all three cell lines, the VRS/DTX-
42.55±9.61 % and 54.24±9.66 % when exposed to LPH nanoparticles demonstrated better performance
VRS and DTX, respectively. Notably, at the same drug compared to the free individual drug or combination
concentration, the cells treated with the VRS/DTX drug treatment. The small size and good internalization
combination were significantly inhibited. To determine could contribute to the superiority of VRS/DTX-LPH.
the synergistic effects of the combination, the data Moreover, interestingly, at a concentration of 1 μM,
were analysed by CalcuSync software. After screening the free combined drugs achieved better activity. This
at various concentrations, the calculated CI for the could be attributable to the sustained release of drugs
SCC-7, MCF-7, and MDA-MB-231 cancer cell lines from the nanoparticles, which caused the effective
were, 0.469, 0.698, and 0.453 (CI <1), respectively, concentration to be achieved much later.
at a VRS/DTX ratio of 2:1, which indicated the
Cellular apoptosis was assayed by flow cytometry
synergistic effect of the VRS and DTX combination.
to determine the changes of cells in the presence or
Furthermore, in comparison with the individual
absence of sample treatments (fig. 4). As shown in
fig. 4, the control group for all cell lines, which were
(A) 120
only exposed to media, showed negligible apoptotic and
100
necrotic cells. In contrast, the apoptotic cell proportion
Cell viability (%)

80 was significantly increased after the treatment of free

60 VRS, free DTX, or VRS/DTX-LPH. In terms of the free
40 drug, VRS and DTX individually induced apoptosis
20 in the treated cells; however, at a combination ratio
0
2:1, the number of observed early and late apoptotic
1 10 20 30 cell was augmented. The data in MCF-7 cells clearly
Drug concentration (μM) showed approximately 47 % for VRS and DTX and
(B) 120 72 % for the VRS/DTX combination. Similar results
100
were obtained in SCC-7 and MDA-MB-231, although
Cell viability (%)

80 the differences were slightly smaller. In another

60 comparison, the apoptotic study also was conducted for
40 different concentrations and incubation times of VRS/
20 DTX-LPH. Among the drug-loaded LPHs, apoptosis
0
was induced at a higher rate with higher incubation
1 10 20 30 time or concentration, which was attributable to the
Drug concentration ( μM) enhancement of intracellular uptake. In comparison
(C) 120 with the free VRS/DTX combination, cancer cells
100 reproduced the apoptosis pattern underwent treatment
Cell viability (%)

of drug-loaded LPH, as indicated by a similar number

80
of apoptotic cells. Those results were consistent with
60
the intracellular uptake and in vitro cytotoxicity studies.
40
Finally, to determine the change in the morphology of
20
cell nuclei that may occur in apoptosis, Hoechst 33342
0
1 10 20 30 staining was used. It is normal to see fairly smooth and
Drug concentration ( μM) homogenous nuclei, as in the case of untreated cells,
Fig. 3: In vitro cytotoxicity in SCC-7, MCF-7, MDA-MB-231 but nuclear fragmentation and apoptotic bodies were
cells
In vitro cytotoxicity of 24 of exposure of blank LPH detected in drug-treated cells (fig. 5). A bright blue
nanoparticles, free DTX, free VTS, VRS/DTX combination, fluorescent condensed nucleus was a feature of cells
and VRS/DTX-LPHs in (A) SCC-7, (B) MCF-7, and (C) MDA- treated with VRS, whereas nuclear fragmentation
MB-231 cells ( ) control; ( ) VRS; ( ) DTX; ( ) VRS/DTX
combination; ( ) VRS/DTX-LPH; data expressed as mean±SD
tended to occur in cells treated with DTX. Both
(n = 8); **p-value <0.01 phenomena simultaneously observed in the cell-treated
479 Indian Journal of Pharmaceutical Sciences May-June 2019
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VRS/DTX
Control VRS DTX combination VRS/DTX-LPH

SCC-7
PI
MCF-7
MDA-MB-231

ANNEXIN V - FITC
Fig. 4: Cellular apoptosis under flow cytometry
Analysis of cellular apoptosis by flow cytometry after treatment for 24 h with 20 μM each of free DTX, free VTS, VRS/DTX
combination and VRS/DTX-LPHs

VRS/DTX
Control VRS DTX combination VRS/DTX-LPH
SCC-7
MCF-7
MDA-MB-231

Fig. 5: Nuclear apoptosis under fluorescent microscopy

Nuclear apoptosis assay using fluorescent microscopy after treatment for 24 h with 2 μM each of free DTX, free VTS, VRS/DTX
combination and VRS/DTX-LPHs nanoparticles

combined drugs and optimized formulation. The that were measured in the experimental data. These
specific mechanism at the molecular level indicated results demonstrated that the successful incorporation
that VRS induced an augment in the expression of of VRS and DTX LPHs demonstrated the potential
acetylated tubulin, which could enhance apoptosis and to overcome drug resistance and also improve the
was also related to DTX activity[11]. anticancer efficacy of chemotherapy.
In summary, a simple drug delivery system for Acknowledgment:
combination chemotherapy was developed. The
nanoparticles of the VRS/DTX-LPH formulation were This research was supported by the Yeungnam
fabricated with a small size, high payload capacity, University research grant in 2018.
and the ability for controlled drug release. The well-
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