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Doxorubicin loaded on chitosan-protamine nanoparticles triggers apoptosis via downregulating Bcl-2 in breast cancer cells
Journal Pre-proof Doxorubicin loaded on chitosan-protamine nanoparticles triggers apoptosis via downregulating Bcl-2 in breast cancer cells Mohamed A. Abd-Elhakeem, Omnia M. Abdel-Haseb, Shaimaa E. Abdel-Ghany, Emre Cevik, Hussein Sabit PII:
S1773-2247(19)31522-9
DOI:
https://doi.org/10.1016/j.jddst.2019.101423
Reference:
JDDST 101423
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 7 October 2019 Revised Date:
14 November 2019
Accepted Date: 26 November 2019
Please cite this article as: M.A. Abd-Elhakeem, O.M. Abdel-Haseb, S.E. Abdel-Ghany, E. Cevik, H. Sabit, Doxorubicin loaded on chitosan-protamine nanoparticles triggers apoptosis via downregulating Bcl-2 in breast cancer cells, Journal of Drug Delivery Science and Technology (2019), doi: https:// doi.org/10.1016/j.jddst.2019.101423. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
1
Doxorubicin Loaded on Chitosan-protamine Nanoparticles Triggers Apoptosis via
2
Downregulating Bcl-2 in Breast Cancer Cells
3
Mohamed A. Abd-Elhakeem1, Omnia M. Abdel-Haseb1, Shaimaa E. Abdel-Ghany2, Emre
4
Cevik3 and Hussein Sabit3†
5
1
6
Science and Technology, P. O. Box 77, Giza, Egypt.
7
2
8
Science and Technology, P. O. Box 77, Giza, Egypt.
9
3
Department of Pharmaceutical Biotechnology, College of Biotechnology, Misr University for
Department of Environmental Biotechnology, College of Biotechnology, Misr University for
Department of Genetics, Institute for Research and Medical Consultations, Imam Abdulrahman
10
Bin Faisal University, P. O. Box 1982, Dammam, 31441 Saudi Arabia.
11
†Corresponding author: [email protected]
12 13
Abstract
14
Cancer-specific drug delivery is a reliable approach to evade undesirable side effects and
15
increase the bioavailability of the drug in tumor cells. In the present study, we treated breast
16
cancer cells MDA-MD-231with doxorubicin (DOX) loaded on chitosan-protamine nanoparticles
17
(CPNPs) to investigate the composite ability to induce apoptosis. CPNPs were prepared and
18
characterized using FTIR spectroscopy, transmission electron microscope, and zeta potential
19
determination. CPNPs showed a drug encapsulation efficiency (EE) of 21%, drug loading
20
capacity (LC) of 3.65% and a particle size of 117 nm. In vitro release study indicated that DOX
21
release from CPNPs-DOX was pH-dependent, where it released with rates 60.10%, 44.15% and
22
25.10% at pH 4.0, 6.8 and 7.4, respectively. Cells were treated with three concentrations (1, 2,
23
and 3 µM) of either free DOX, doxorubicin loaded on CPNPs (CPNPs-DOX), or empty carrier
24
for 48 h. Cell viability was assessed using MTT and trypan blue assays. Meanwhile, apoptosis
25
rate using PI/Annexin V-FITC staining cell cycle analysis were performed using PI staining-
26
based flow cytometry. MTT and trypan blue assays showed a significant decrease in the
27
viability/count upon treating cells with DOX-CPNPs. Flow cytometry data revealed an arrest of
28
the breast cancer cell at G2/M (47.18%) in the CPNPs-DOX treatment. Quantitative real time
29
PCR analysis showed that CPNPs-DOX treatment has significantly downregulated Bcl-2
30
compared with free DOX treatment and control. These results indicate the efficiency of using
31
CPNPs as a drug carrier for DOX in treating breast cancer cells, however, these conclusion needs
32
further investigation.
33
Keywords: Breast cancer; doxorubicin; Adriamycin; Chitosan Nanoparticle; Protamine; Bcl-2.
34
1. Introduction
35
Breast cancer (BC) is the most common cancer among women, affecting 2.1 million women
36
annually, with the highest number of deaths from cancer. By the end of 2019, an estimated
37
268,600 new cases of breast cancer are expected to be diagnosed in the US, with 41,760 are
38
expected to die [1]. BC incidence rates are higher among women in developed countries. The 5
39
primary risk factors of breast cancer include age [2], high hormone level [3], race [4], economic
40
status [5], and iodine deficiency in diet [6]. Current breast cancer treatment modalities include
41
surgery, chemotherapy, and radiotherapy or a combination of these options [7]. All these
42
treatments cause damage to healthy tissues or incomplete eradication of cancer.
43
Though conventional chemotherapies such as DOX has proved successful to some extent, some
44
drawbacks are there including adverse side effects, poor bioavailability, non-specific targeting,
45
low therapeutic indices, development of multiple drug resistance [8]. Chemotherapeutic drugs
46
are delivered primarily into the cytoplasm, where it enters the nucleus through the nuclear pores.
47
Based on its size and bioavailability, some drugs are not transferred to the nucleus, resulting in
48
poor therapeutic efficiency [9, 10]. To improve the effectiveness and safety of cancer
49
chemotherapy, drug delivery systems such as microsphere, nanoparticles, and liposomes can be
50
used [11].
51
Nanotherapeutics is a fast-growing cancer research field aimed at resolving numerous limitations
52
of conventional drug delivery systems. The non-specificity of cancer chemotherapy results in the
53
targeting of rapidly dividing normal cells [12]. Nanotherapeutics help to overcome some of the
54
drawbacks of chemotherapies such as lack of selectivity, multidrug resistance and lack of
55
bioavailability [13]. In addition, the use of nanotechnology-based therapies leads to a lower
56
patient risk and an enhanced rate of survival [14]. Drug nanocarriers are colloidal systems with
57
sizes below 500 nm. These nanocarriers, owing to their high surface area to volume ratio and
58
sustained drug release, can improve drug pharmacokinetics and bioavailability, and decrease
59
drug toxicity and adverse side effects, making it a unique drug delivery system. In general, the
60
overall goal of using nanocarriers in the delivery of drugs is to effectively treat a disease with
61
minimal side effects [15]. Chitosan is a polysaccharide of biological origin, where it is used in a
62
broad range of medical applications due to its biocompatibility and biodegradability. It has been
63
used as a drug carrier, gene delivery tool, and as an immunoadjuvant for vaccines [16].
64
Protamines are a diverse family of small arginine-rich proteins that are synthesized in the late-
65
stage spermatids of many animals and plants and bind to DNA, condensing the spermatid
66
genome into a genetically inactive state [17]. Protamine is nuclear proteins containing a nuclear
67
localization signal (NLS), which is an amino acid sequence consisting of one or more short
68
sequences of positively charged lysine or arginine that could deliver proteins and chemical drugs
69
to the nucleus by forming the nuclear pore complex, [18] thus improving the uptake efficiency of
70
exogenous substances into the nucleus [19].
71
Doxorubicin is a metabolite produced by Streptomyces peucetius. DOX is used as an
72
antineoplastic agent to treat fluid and solid cancer, including leukemia breast cancer.
73
Unfortunately, the use of DOX is associated with toxicity that might lead to extravasation,
74
nausea, vomiting, hematopoietic suppression, alopecia, and cardiotoxicity [20].
75
Apoptosis is the main resistance mechanism against tumor development and it is fundamental for
76
cellular homeostasis [21]. B-cell lymphoma 2 (Bcl-2) is the primary member of the Bcl-2
77
family of regulatory proteins that regulate the molecular mechanisms of apoptosis by its dual
78
function of inhibiting or inducing apoptosis [22]. Members of this family include Bcl-2-
79
associated protein X (Bax) that induces and accelerates cell death when present as Bax/Bax
80
homodimer. Whereas, Bcl-2 and B-cell lymphoma extra-large (Bcl-xL) exert antiapoptotic action
81
when the heterodimers Bcl-2/Bax or Bcl-xL/Bax are formed [23, 24]. The interaction between
82
Bcl-2 family members determines the fate of the cell for death or survival [25].
83
The present study aimed at evaluating the antitumor activity of CPNPs-loaded DOX against
84
breast cancer MDA-MD-231.
85 86
2. Materials and Methods 2.1.Biological materials
87
MDA-MD-231 breast cancer cell line was purchased from the Holding Company for Biological
88
Product and Vaccines (VACSERA), Giza, Egypt. DOX and dimethyl sulfoxide (DMSO) were
89
purchased from Sigma-Aldrich (Germany).
90
2.2.Preparation of chitosan-protamine nanoparticles
91
The preparation of CPNPs was carried out according to Yue et al. [26], where 20 mg of chitosan
92
(Deacetylation degree 95%, molecular weight 80 kDa, Sigma Aldrich) was dissolved in 100 mL
93
of 0.1% acetic acid. pH 4.7 was adjusted using sodium hydroxide. Protamine (5 mg) was
94
dispersed in 4 mL ethanol, chitosan and protamine solutions were mixed for 30 min. by magnetic
95
stirring. 20 mg sodium tripolyphosphate (1mg/mL) was added drop by drop to the formed
96
mixture. The obtained NPs were centrifuged at 12,000 rpm for 15 min. and washed three times
97
with distilled water to ensure complete removal of non-encapsulated drug. The pellets were
98
freeze-dried and store at -4 oC till use.
99
2.3.Encapsulation of doxorubicin in chitosan-protamine nanoparticles
100
The preparation of DOX loaded on CPNPs were performed by the same method with the
101
addition of 3 mg DOX to the chitosan -protamine mixture before the dropping of
102
tripolyphosphatesodium (TPP).
103
2.4.Characterization of nanomaterial
104
CPNPs and DOX- CPNPs were suspended in phosphate buffer (pH = 7.4), and their sizes, zeta
105
potentials, and polydispersity index (PDI) were analyzed by Dynamic Light Scattering (DLS,
106
Zeta sizer Nano ZS, Malvern Instruments, Worcestershire, UK) at room temperature. The surface
107
morphology of NPs was observed by transmission electron microscopy (JEOL, JAM-2100-HR-
108
EM). Finally, the infrared spectra of free DOX, CPNPs and DOX- CPNPs were measured by a
109
Fourier-transform infrared (FT-IR) spectroscopy analyzer (Model JASCO FTIR-6100) within the
110
scanning range 4000–400 cm-1.
111 112
2.5. Determination of Encapsulation efficiency and drug loaded capacity
113
Five milligrams of freeze-dried DOX-CPNPs were vigorously vortexed in 1 mL methanol for 30
114
min. Then, the nanoparticles suspension was centrifuged at 12,000 rpm for 10 min. The
115
supernatant was used to determine the DOX content by measuring absorption at 480 nm by
116
(UV/VIS spectrophotometer, Shimadzu UV1800). Measurements were carried out three times,
117
then encapsulation efficiency (EE%) and loading capacity (LC %) were calculated by the
118
following equation: % = % =
119
ℎ
100
!
100
2.6.In vitro drug release study
120
Twenty milligrams of freeze-dried DOX-CPNPs were placed into a regenerated cellulose
121
dialysis bag (MWCO, 8000–14000, Sigma, St. Louis, MO). The closed bag was immersed into
122
50 mL of release medium (PBS pH = 5.0, 6.8, 7.4, 0.1 mol/L). The dialysis bag was incubated at
123
37oC under gentle agitation. At specific time intervals, 1 mL of release medium was removed for
124
DOX determination using spectrophotometric analysis at 480 nm. Each batch of experiments
125
was performed in triplicate. Cumulative percentage of DOX released was obtained by dividing
126
the cumulative amount of DOX released at each sampling time point (Mt) to the initial weight of
127
the CPNPs-DOX in the sample (M0), as presented in the following equation:
Cumulative release percentage = 1
Mt X 100 456 M0 4
128 129
2.7.Cell lines maintenance and drug treatment
130
The human triple-negative breast cancer cell line MDA-MB-231 was maintained on DMEM
131
media supplemented with antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin, Life
132
Technologies, NY, USA) and 10% FBS (Sigma-Aldrich). Cells were cultured in incubator at 37
133
˚C with 5% CO2 24 h prior treatment with three concentrations of free DOX concentration (1, 2,
134
and 3 µM in 0.05% DMSO), CPNPs or CPNPs-DOX (1mg/mL, 2 mg/mL, 5 mg/mL) at 70%
135
confluence for 48 h.
136
2.8.Trypan blue assay
137
Viable MDA-MB-231 cells were cultured in a 12-well tissue culture plate and treated with
138
different concentrations of free DOX, CPNPs, or CPNPs-DOX for 48 h. To assess the viability,
139
adherent cells were dislodged with 0.25% EDTA trypsin (Sigma-Aldrich). Cells were harvested
140
at low speed centrifugation and resuspended in 1 mL of normal growth medium. Equal volume
141
of cells and 0.4% trypan blue dye was mixed and left for 3 min at room temperature and then
142
loaded on hemocytometry slide. The viable and dead cells were counted under light microscope
143
(Olympus, BX43). Four readings were taken for each well, and the average was calculated. The
144
number of viable cells was calculated as follows:
145 146
Cell count = Average cell counts for the four readings x 2 x 104. 2.9.Cytotoxicity assay
147
Cell viability of MDA-MB-231 was determined by using 3-(4,5- dimethylthiazol-2-yl)-2,5-
148
diphenyl-tetrazolium bromide (MTT). Cells were seeded on 12-well plate with concentration of
149
5 x104 cells/well. Seeded wells were treated for 48 h with designated concentrations of DOX in
150
triplicates, and the rest of wells remained untreated to serve as control. After incubation, the
151
culture medium was replaced with 150 µL fresh media and 50 µL MTT (5 mg/mL in PBS), and
152
the plate was incubated for 4 h at 37 ˚C in a humidified atmosphere with 5% CO2. The developed
153
formazan crystals were dissolved by adding 200 µL of DMSO. The plate was incubated for 30
154
min. at 37 ˚C and then the optical density was determined at 550 nm using a spectrophotometric
155
microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).
156
2.10.
Cell cycle analysis
157
To assess the changes in the cell cycle after DOX treatment, 4 × 104 cells per well in a 12-wells
158
plate were seeded and treated with free DOX, CPNPs or CPNPs-DOX as previously described.
159
Cells were collected 48 h post-treatment at 600 rpm for 5 min. in cold 70% ethanol in PBS.
160
Cells, then, were centrifuged again at 600 rpm for 5 min. and left at 4 ºC for 2 h. Cells were
161
treated with 50 µg/mL PI, 0.1% Triton X-100 and 50 µg/mL RNAse for 25 min. and incubated at
162
room temperature in a dark place. The PI fluorescence was read on a FACScan flow cytometer
163
(BD FACSCalibur™). Data were analyzed to show the cell cycle distribution of the treated and
164
untreated cells.
165
2.11.
Apoptosis detection
166
MDA-MB-231 breast cancer cells were cultured in 12-well tissue culture plate and were treated
167
with free DOX, empty CPNPs, or CPNPs-DOX for 48 h. The apoptotic cells were identified
168
using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. Briefly,
169
treated and control cells were harvested and resuspended in 100 µL Annexin V binding buffer
170
and 5 µL Annexin V Alexa Fluor 488. The mixture was incubated for 15 min. in a dark place. PI
171
(4 µL) diluted in 1x Annexin V binding buffer (1: 10) was added and the mixture and incubated
172
again for 15 min. in a dark place.
173
Annexin V binding buffer (500 µL) was added to wash the Annexin/PI stained cells. Annexin/PI
174
staining was visualized on flow cytometer (BD FACSCalibur™). Annexin V-FITC binding was
175
analyzed at Ex = 488 nm; Em = 530 nm using FITC signal detector (FL1) and PI staining by the
176
phycoerythrin emission signal detector (FL2). 2.12.
177
RNA extraction and cDNA synthesis
178
Total RNA was extracted from control, free DOX-treated, empty CPNPs-treated, and CPNPs-
179
DOX-treated cells using RNA Isolation System (Qiagen, GmbH, Germany). RNA Quality was
180
evaluated by agarose gel electrophoresis and RNA concentration was measured by A260/A280
181
using NanoDrop 1000 Spectrophotometer (Wilmington, DE, USA). To synthesize cDNA, 5 µg
182
of the extracted RNA was mixed with 1µg random 6-mer primers and 1µM deoxyribonucleotides
183
(dNTP), 10 units of M-MLV SuperScript II Reverse Transcriptase (Invitrogen). The mixture was
184
incubated for 60 min. at 42 °C. 2.13.
185
Gene expression analysis
186
Quantitative real time PCR was used to amplify B-cl-2 gene. Primer sequences used in this study
187
are
188
CAATCCTCCCCCAGTTCACC-3'. Primers were generated using Primer-BLAST tool, NCBI.
189
About 100 ng (2 µL) of cDNA was mixed with 12.5 µL Cyber Green master mix, 10 pM (1.5
190
µL) of each of the primers, and the final volume was brought to 25 µL with molecular biology-
Bcl-2
forward:
5'-TCTCATGCCAAGGGGGAAAC-3'
and
Bcl-2
reverse:
5'-
191
grade water. The thermal profile was as follows: pre-PCR heating for 5 min. at 95 ºC, then 35
192
cycles of 94 ºC for 40 sec., 56 ºC for 45 sec., 72 ºC for 50 sec. followed by a final extension step
193
of 72 ºC for 10 min. All reactions were performed in triplicates on StepOne Plus thermal cycler
194
(Applied Biosystems, UK). 2-∆∆CT method was used to calculate the fold change in Bcl-2 gene
195
expression.
196
2.14.
Statistical analysis
197
Results of all experiments were presented as mean ± standard error (SE) in triplicate
198
experiments. All analyses were performed using the student's t-test to compare control with
199
treatment. P<0.05 was considered significant.
200 201
3. Results 3.1.Characterization of nanomaterial
202
As shown in (Fig. 1) the size distribution of CPNPs ranged from 90 to 800 nm with main peak at
203
163.50 nm and polydispersity index (PDI) of 0.512. While the CPNPs-DOX showed relatively
204
narrow particle size distribution ranged from 90 to 200 nm with main peak at 117 nm and PDI
205
value of 0.653. The zeta potential was 5.43 mV and 30.3 mV for CPNPs and CPNPs-DOX,
206
respectively (Fig. 1). The positive zeta potential was due to the residual amine groups. TEM
207
analysis revealed that the obtained nanoparticles were spherical and regular in shape and range in
208
size 100-150 nm (Fig. 2A). Finally, FTIR spectra of DOX, CPNPs, and CPNPs-DOX has several
209
bands as compared to those of free DOX or CPNPs. In brief, the FTIR spectrum of DOX shows
210
characteristic peaks at 3080 (C–H), 1730 (C=O) cm-1. These peaks are present in the FTIR
211
spectrum of CPNPs-DOX with small shift at 3050 (C–H), 1750 (C=O) cm-1. These results
212
indicate the successful loading of DOX in CPNPs (Fig. 2B).
213
3.2.Encapsulation efficiency and drug loading capacity
214
The encapsulation efficiency and drug loaded capacity were calculated according
215
aforementioned equations (materials and Methods section). Data obtained showed encapsulation
216
efficiency of 21% and the drug loaded capacity of 3.65%. According to these results, we
217
determined the concentrations of the CPNPs and CPNPs-DOX applied to the cells; 2.77 µg, 5.54
218
µg and 8.31µg, which contain the equivalent concentrations of the free DOX (1 µM, 2 µM and 3
219
µM).
220
Fig. 1
221
Fig. 2
222 223
3.3.In vitro release study
224
In the present study, we evaluated the release profile of DOX from CPNPs at three different pH
225
values. A quick release of DOX was seen in the first 6 h (approximately 40%, 28% and 16% of
226
the drug were released at pH 5.0, 6.8 and 7.4, respectively) then a relatively slow and sustained
227
release was observed in the following hours. In general, the amount of drug released at acidic
228
condition (pH 5.0) was greater than in other pH values (Fig. 3).
229
230
231
Fig. 3
232
3.4.Cytotoxicity assay
233
Cytotoxicity of the MDA-MB-231 breast cancer cells treated with free DOX, CPNPs, or CPNPs-
234
DOX was assessed by MTT assay and trypan blue dye exclusion test. Cells were incubated with
235
the drugs for 48 h and then harvested to assess viability. MTT assay results indicated that
236
CPNPs-DOX significantly reduced the overall cell viability compared to CPNPs and the free
237
DOX. The best CPNPs-DOX concentration was 3 µg/mL, where it resulted in 2% cell viability,
238
whereas free DOX at the same concentration resulted in 35% cell viability (Fig. 4).
239
Fig. 4
240 241 242
3.5.Cell cycle analysis
243
DNA content and apoptosis rate were analyzed in MDA-MB-231 cells treated with different
244
concentrations of encapsulated or free DOX. Flow cytometry data showed that CPNPs-DOX
245
(3µg/mL) yielded the highest percentage of cell growth arrest at G2/M stage compared to control
246
and free DOX (Fig. 5). Apoptosis was also detected in the treated and untreated cells using
247
PI/Annexin V-FITC staining. Data revealed that the percentages of cells undergone late
248
apoptosis were 1.63%, 14.26%, 11.11%, and 28.72% in control, free DOX treatment, CPNPs
249
treatment, and CPNPs-DOX treatment, respectively (Fig. 6).
250 251
Fig. 5
252
Fig. 6
253 254
3.6.Gene expression analysis
255
Bcl-2 is believed to be an apoptosis suppressor gene, and the upregulation of the protein in
256
cancer cells may interrupt the onset of apoptosis. In the present study Bcl-2 was downregulated
257
in breast cancer cells treated with different concentrations of free DOX, CPNPs, and CPNPs-
258
DOX. qPCR data showed that the level of Bcl-2 expression was negatively correlated with
259
elevation of CPNPs-DOX concentration, where it reached 0.04% in the highest concentration (3
260
µg/mL) relative to untreated cells and house-keeping gene (Fig. 7).
261
Fig. 7
262 263 264 265
4. Discussion 4.1.In vitro release study
266
Naturally, pH is gradient in the endosomal or lysosomal of tumor cells (5.0–6.5) and tumor
267
microenvironment (6.5–7.2) [27]. pH-sensitive nanoparticles might actively release drug at the
268
tumor site or in the endosomal or lysosomal of target tumor cells, which can enhance the
269
antitumor activity, as well as reduce potential damage to normal cells.
270
The significant decrease in the count of viable cells exhibited by CPNPs-DOX treatment might
271
indicate that the carrier CPNPs enhanced the bioavailability of DOX. Interestingly, the empty
272
CPNPs showed no toxicity in concentrations up to 5 mg/mL, which proved the biocompatible
273
nature of this nanocarrier. Taking these two findings together, we can conclude that the severe
274
decrease in cell viability is attributed mainly to DOX released from CPNPs. Other studies
275
indicated the obtained profile although in other types of cancers. Fang et al., [28] reported a
276
significantly higher intracellular DOX concentrations and more apoptotic induction in lymphoma
277
cells treated with DOX-loaded nanocarrier. DOX-loaded CNTs can be released in a sustained
278
manner and exert an effective antitumor activity in cervical cancer cells, making it potentially
279
promising therapeutic option in cancer treatment [29]. Our in vitro release experiment revealed
280
that releasing DOX from CPNPs-DOX was pH-dependent, where 40%, 28% and 16% of the
281
drug were released at pH values of 5.0, 6.8 and 7.4, respectively. When DOX was loaded on
282
MgO nanoflakes, it exhibited releasing rates of 90.2%, 50.5%, and 10% at pH values of 3, 5.0,
283
and 7.2, respectively [30]. These data might indicate the superiority of MgO nanoflakes as a
284
nanocarrier over CPNPs. Das et al. [31] indicated that DOX-loaded nanoceria exhibited higher
285
cellular uptake and drug release rates compared with free DOX, and this release is enhanced
286
under reductive acidic conditions (pH 5.0, 10mM glutathione) than under physiological
287
conditions (pH 7.4). Besides its enhanced bioavailability, DOX-loaded nanocarriers (such as
288
DOX-Fe-PDA/FA-PEG) can also trigger intracellular ROS overproduction, thereby enhancing
289
its therapeutic effect on breast cancer [32]. Other approaches use chitosan nanobubbles (NBs) as
290
a carrier for delivering DOX in cancer cells. Zhou et al. [33] reported the efficiency of DOX-NBs
291
to release DOX in MCF-7 breast cancer cells.
292
4.2.Encapsulated DOX and cell viability
293
Generally, the size and surface properties of NPs have a vital role in drug release, in vivo
294
pharmacokinetics, and cellular uptake [34]. The tiny size allows nanoparticles to circulate
295
through the microvascular bed of a tumor and extravasate into the perivascular space by
296
convective transport through the endothelium and retained at the site [35]. In the present study,
297
the obtained DOX- CPNPs had a nano size and relatively small PDI value that indicated that the
298
size distribution of DOX-NPs was narrow.
299
Breast cancer cells were treated with different doses of free DOX, CPNPs or CPNPs-DOX for 48
300
h to elucidate the efficiency of encapsulated DOX in inducing apoptosis via downregulating Bcl-
301
2. MDA-MB-231 breast cancer cell viability assays revealed a very significant decrease (p =
302
0.00026) in the percentage of viable cells treated with CPNPs-DOX (2%) at the highest
303
concentration (3 µg/mL) compared to free DOX (p = 0.002) and empty CPNPs (p = 0.08). It has
304
been reported that DOX-loaded PLA-TPGS nanoparticles (with about 100 nm in size) also
305
exhibited higher cytotoxicity and cellular uptake on both HeLa and HT29 cells [36]. Meanwhile,
306
other composites such as DOX-CeO2 also showed higher cell proliferation inhibition in ovarian
307
cancer compared with free DOX. A recent study indicated that the free DOX resulted in 35% and
308
89% cytotoxicity when MCF-7 cells treated with 10 µM and 2500 µM, respectively [37].
309
Although its benefits, using DOX in free form requires higher doses, which can be harmful to the
310
normal cells. Thus, loading DOX to a nanocarrier represents a safe way to treat cancer.
311
4.3.Apoptosis induction
312
Programmed cell death occurs as a cellular response to either external or internal stimuli. Thus,
313
regulation of this process is crucial for normal growth and homeostasis. Inducing apoptosis,
314
especially via disrupting the apoptotic machinery, is a proved way to treat cancer. In the present
315
study, we treated breast cancer cells with different concentrations of free DOX and DOX-loaded
316
CPNPs for 48 h. Results indicated that DOX-loaded CPNPs treatment (eq. to 3 µg/mL) has
317
resulted in a significant increase (p = 0.049) in the percentage of apoptosis (28.72%) compared
318
with control (1.63%), making this composite a potential treatment for breast cancer. Several
319
studies indicated the efficiency of loading DOX on nanocarrier in apoptosis induction. Siddharth
320
et al. [38] reported that DOX-loaded-PLGA-PVA-NP enhanced the uptake of DOX in MCF-7-
321
DOX-R cells and caused apoptosis by increasing both apoptotic nuclei and Bax/Bcl-xL ratio.
322
DOX-loaded FA-Se induced apoptosis rates (24.77%) in cervical cancer cells compared with the
323
cells treated with free DOX (10.48%). These data indicated that DOX-loaded FA-Se could
324
augment the antitumor activity of DOX in cervical cancer [39]. Furthermore, DOX-loaded CNPs
325
conjugated with FA caused enhanced release of cytochrome c as well as the activation of
326
downstream caspases to assist apoptosis in Y-79 retinoblastoma cells, which may provide a drug
327
delivery system (e.g. DOX) for the treatment of retinoblastoma [40]. In conclusion, nanocarriers-
328
mediated drug delivery offers a safe, fast, and reliable approach to augment the available
329
strategies to combat cancer.
330
4.4.Cell cycle analysis
331
In the present investigation, we analyzed the breast cancer cells after being treated with DOX or
332
CPNPs-DOX composite using PI staining. Data obtained indicated that CPNPs-DOX treatment
333
has resulted in the highest percentage of pre-G1 (28.72%) compared with control (1.53%), free
334
DOX (14.26%), and CPNPs (11.11%), which represents the effect of this composite in triggering
335
cell death. Other composites such as DOX-loaded FA-Se showed greater activity to induce
336
apoptosis in cervical cancer cells compared with free DOX. Pre-G1 apoptosis peak was 29.58%
337
and 13.69% when cells were treated with DOX-loaded FA-Se and free DOX, respectively [39].
338
Furthermore, treating MCF-7 breast cancer cells with DOX- loaded- PLGA-PVA-NPs has
339
resulted in a notable increase of Sub-G1, indicating the efficiency of this composite in inducing
340
apoptosis [38]. Meanwhile, our prepared composite arrested breast cancer cells at G2/M phase
341
(47.18%), which indicate its antiproliferative effect, and hence its efficacy as a therapeutic agent
342
for cancer. Neither DOX nor CPNPs did not exhibit this effect. MCF-7 treated with DOX-loaded
343
MagAlg (at 5 µg/mL and 0.5 µg/mL) showed an increase in the number of cells in G2/M phase
344
(20%) [41]. This might indicate that CPNPs-DOX is more efficient than DOX-loaded MagAlg in
345
inhibiting breast cancer proliferation.
346
4.5.Gene expression analysis
347
We analyzed the expression of Bcl-2 gene in treated and untreated cells. Results indicated that
348
DOX-loaded CPNPs treatment has resulted in a significant decrease in Bcl-2 expression (87%)
349
compared with CNPNs (72%) and free DOX (84%). Bcl-2 serves as an inhibitor of the intrinsic
350
(mitochondrial) apoptosis pathway, where it blocks Bid and Bax to prevent the release of
351
cytochrome c, that activates caspase cascade. Thus, downregulation of Bcl-2 is the main
352
triggering factor for the cells to undergo apoptosis [42]. It was indicated that DOX decreases the
353
anti-apoptotic Bcl-xL and increases pro-apoptotic Bax mRNA levels [43], leading to the
354
execution of apoptosis. Increasing the bioavailability of DOX within tumor might help
355
completely remove these cells. Other studies indicated the downregulation of Bcl-2 in different
356
cells including MDA-MB-231 breast cancer cells, MTLn3 adenocarcinoma cells treated with
357
DOX [44-46].
358
5. Conclusion
359
The main drawback of chemotherapy is the decreased bioavailability within cancer cells. In the
360
present investigation, novel protamine-chitosan pH-sensitive DOX nanoparticles were developed
361
and characterized using different tools, including TEM, FT-IR, zeta potential and size. We
362
treated MDA-MB-231 breast cancer cells with different concentrations of this composite along
363
with free DOX and empty carrier. Our data indicated that treating cells has resulted in a
364
significant decrease in the cell viability and cell count. Furthermore, apoptosis rate and cell cycle
365
phase distributions were also analyzed. All obtained data showed the efficacy of the DOX-loaded
366
CPNPs in inducing apoptosis and arresting cells at G2/M phase, indicting the antiproliferative
367
activity of the composite compared with free DOX and the empty carrier. This study concludes
368
that DOX-loaded CPNPs represents an efficient combination to enhance the chemotherapeutic
369
action of DOX in treating breast cancer.
370 371
Author participation
372
MA: conceptualized the idea, OM and SEA: conducted the experimental work, EC: prepared
373
drawings and illustrations, HS: wrote the manuscript.
374
Conflict of interest
375
The authors declare no conflict of interests
376
Acknowledgement
377
This work received no fund from any funding bodies
378 379
References
380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396
Figures and Ligands
397
Fig. 1
398 399 400 401 402 403 404 405
Fig. 1: Analysis of composite zeta size and zeta potential. A: CPNPs zeta size, B: CPNPs-DOX
406
zeta size, C: CPNPs zeta potential, and D: CPNPs-DOX zeta potential.
407
408
409
Fig. 2
A 410 411 412 413 414 415
B
416
Fig. 2: A: TEM image of CPNPs-DOX reveals the spherical shape of the composite that range in
417
size 100-150 nm. B: FTIR spectroscopic analysis reveals three distinct chemical composition of
418
a: CPNPs, b: free DOX, and c: CPNPs-DOX.
419 420 421 422 423 424 425
426
427 428 429 430 431 432 433
Fig. 3
434
435
436
Fig. 3: In vitro release profile of DOX from CPNPs at three different pH values. A quick release
437
of DOX was seen in the first 6 h with approximately 40%, 28% and 16% of the drug was
438
released at pH 5.0, 6.8 and 7.4 respectively. In general, the amount of drug released at acidic
439
condition (pH 5.0) was greater than other pH values.
440 441 442 443 444 445
446
Fig. 4
447 A 448 449 450 451 452
453
B
454 455 456 457 458 459 460 461 462
Fig. 4: Cell viability and cell count. A: MTT assay revealed a significant decrease in the breast
463
cancer cell viability after being treated with CPNPs-DOX compared with free DOX and CPNPs.
464
B: Cell count using trypan blue indicated a significant decrease in the count of cells treated with
465
the prepared composite compared with free DOX and CPNPs.
466
467
Fig. 5
468 469 470 471 472 473 474 475 476 477 478 479 480 481 482
Fig. 5: Cell cycle distribution and DNA content. The distribution of cell cycle phases after
483
treating breast cancer cells with DOX, CPNPs, and DOX-loaded CPNPs. Data revealed an arrest
484
at G2/M phase when cells treated with the prepared composite CPNPs-DOX for 48 h at 3 µM.
485
486
Fig. 6 487 488 489
490 491 492 493 494 495 496
Fig. 6: Apoptosis detection. MDA-MB-231 breast cancer cells were treated with DOX, CPNPs,
497
and DOX-loaded CPNPs for 48 h at 3 µM. Annexin-V FT-IC/PI staining indicated a significant
498
increase in the apoptosis rate in cells treated with DOX-loaded CPNPs compared with those
499
treated with frees DOX or CPNPs.
500
501
Fig. 7
502 503 504 505 506 507
508 509 510 511 512 513 514 515 516
Fig. 7: Gene expression analysis of Bcl-2. Treated and untreated cells were subjected to real time
517
PCR to assess the changes in Bcl-2 gene expression as a result of different treatment. Data
518
indicated that cells treated with DOX-loaded CPNPs showed a significant reduction in the
519
expression of Bcl-2 compared with cells treated with either free DOX or empty carrier.
520
521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565
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Conflict of interest All the authors of: “Doxorubicin Loaded on Chitosan-protamine Nanoparticles Triggers Apoptosis via Downregulating Bcl-2 in Breast Cancer Cells” Mohamed Abdel-Hakeem, Omnia Magdy, Shaimaa E. Abdel-Ghany, Emre Cevik and Hussein Sabit3 declare no conflict of interests.
S1773-2247(19)31522-9
DOI:
https://doi.org/10.1016/j.jddst.2019.101423
Reference:
JDDST 101423
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 7 October 2019 Revised Date:
14 November 2019
Accepted Date: 26 November 2019
Please cite this article as: M.A. Abd-Elhakeem, O.M. Abdel-Haseb, S.E. Abdel-Ghany, E. Cevik, H. Sabit, Doxorubicin loaded on chitosan-protamine nanoparticles triggers apoptosis via downregulating Bcl-2 in breast cancer cells, Journal of Drug Delivery Science and Technology (2019), doi: https:// doi.org/10.1016/j.jddst.2019.101423. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
1
Doxorubicin Loaded on Chitosan-protamine Nanoparticles Triggers Apoptosis via
2
Downregulating Bcl-2 in Breast Cancer Cells
3
Mohamed A. Abd-Elhakeem1, Omnia M. Abdel-Haseb1, Shaimaa E. Abdel-Ghany2, Emre
4
Cevik3 and Hussein Sabit3†
5
1
6
Science and Technology, P. O. Box 77, Giza, Egypt.
7
2
8
Science and Technology, P. O. Box 77, Giza, Egypt.
9
3
Department of Pharmaceutical Biotechnology, College of Biotechnology, Misr University for
Department of Environmental Biotechnology, College of Biotechnology, Misr University for
Department of Genetics, Institute for Research and Medical Consultations, Imam Abdulrahman
10
Bin Faisal University, P. O. Box 1982, Dammam, 31441 Saudi Arabia.
11
†Corresponding author: [email protected]
12 13
Abstract
14
Cancer-specific drug delivery is a reliable approach to evade undesirable side effects and
15
increase the bioavailability of the drug in tumor cells. In the present study, we treated breast
16
cancer cells MDA-MD-231with doxorubicin (DOX) loaded on chitosan-protamine nanoparticles
17
(CPNPs) to investigate the composite ability to induce apoptosis. CPNPs were prepared and
18
characterized using FTIR spectroscopy, transmission electron microscope, and zeta potential
19
determination. CPNPs showed a drug encapsulation efficiency (EE) of 21%, drug loading
20
capacity (LC) of 3.65% and a particle size of 117 nm. In vitro release study indicated that DOX
21
release from CPNPs-DOX was pH-dependent, where it released with rates 60.10%, 44.15% and
22
25.10% at pH 4.0, 6.8 and 7.4, respectively. Cells were treated with three concentrations (1, 2,
23
and 3 µM) of either free DOX, doxorubicin loaded on CPNPs (CPNPs-DOX), or empty carrier
24
for 48 h. Cell viability was assessed using MTT and trypan blue assays. Meanwhile, apoptosis
25
rate using PI/Annexin V-FITC staining cell cycle analysis were performed using PI staining-
26
based flow cytometry. MTT and trypan blue assays showed a significant decrease in the
27
viability/count upon treating cells with DOX-CPNPs. Flow cytometry data revealed an arrest of
28
the breast cancer cell at G2/M (47.18%) in the CPNPs-DOX treatment. Quantitative real time
29
PCR analysis showed that CPNPs-DOX treatment has significantly downregulated Bcl-2
30
compared with free DOX treatment and control. These results indicate the efficiency of using
31
CPNPs as a drug carrier for DOX in treating breast cancer cells, however, these conclusion needs
32
further investigation.
33
Keywords: Breast cancer; doxorubicin; Adriamycin; Chitosan Nanoparticle; Protamine; Bcl-2.
34
1. Introduction
35
Breast cancer (BC) is the most common cancer among women, affecting 2.1 million women
36
annually, with the highest number of deaths from cancer. By the end of 2019, an estimated
37
268,600 new cases of breast cancer are expected to be diagnosed in the US, with 41,760 are
38
expected to die [1]. BC incidence rates are higher among women in developed countries. The 5
39
primary risk factors of breast cancer include age [2], high hormone level [3], race [4], economic
40
status [5], and iodine deficiency in diet [6]. Current breast cancer treatment modalities include
41
surgery, chemotherapy, and radiotherapy or a combination of these options [7]. All these
42
treatments cause damage to healthy tissues or incomplete eradication of cancer.
43
Though conventional chemotherapies such as DOX has proved successful to some extent, some
44
drawbacks are there including adverse side effects, poor bioavailability, non-specific targeting,
45
low therapeutic indices, development of multiple drug resistance [8]. Chemotherapeutic drugs
46
are delivered primarily into the cytoplasm, where it enters the nucleus through the nuclear pores.
47
Based on its size and bioavailability, some drugs are not transferred to the nucleus, resulting in
48
poor therapeutic efficiency [9, 10]. To improve the effectiveness and safety of cancer
49
chemotherapy, drug delivery systems such as microsphere, nanoparticles, and liposomes can be
50
used [11].
51
Nanotherapeutics is a fast-growing cancer research field aimed at resolving numerous limitations
52
of conventional drug delivery systems. The non-specificity of cancer chemotherapy results in the
53
targeting of rapidly dividing normal cells [12]. Nanotherapeutics help to overcome some of the
54
drawbacks of chemotherapies such as lack of selectivity, multidrug resistance and lack of
55
bioavailability [13]. In addition, the use of nanotechnology-based therapies leads to a lower
56
patient risk and an enhanced rate of survival [14]. Drug nanocarriers are colloidal systems with
57
sizes below 500 nm. These nanocarriers, owing to their high surface area to volume ratio and
58
sustained drug release, can improve drug pharmacokinetics and bioavailability, and decrease
59
drug toxicity and adverse side effects, making it a unique drug delivery system. In general, the
60
overall goal of using nanocarriers in the delivery of drugs is to effectively treat a disease with
61
minimal side effects [15]. Chitosan is a polysaccharide of biological origin, where it is used in a
62
broad range of medical applications due to its biocompatibility and biodegradability. It has been
63
used as a drug carrier, gene delivery tool, and as an immunoadjuvant for vaccines [16].
64
Protamines are a diverse family of small arginine-rich proteins that are synthesized in the late-
65
stage spermatids of many animals and plants and bind to DNA, condensing the spermatid
66
genome into a genetically inactive state [17]. Protamine is nuclear proteins containing a nuclear
67
localization signal (NLS), which is an amino acid sequence consisting of one or more short
68
sequences of positively charged lysine or arginine that could deliver proteins and chemical drugs
69
to the nucleus by forming the nuclear pore complex, [18] thus improving the uptake efficiency of
70
exogenous substances into the nucleus [19].
71
Doxorubicin is a metabolite produced by Streptomyces peucetius. DOX is used as an
72
antineoplastic agent to treat fluid and solid cancer, including leukemia breast cancer.
73
Unfortunately, the use of DOX is associated with toxicity that might lead to extravasation,
74
nausea, vomiting, hematopoietic suppression, alopecia, and cardiotoxicity [20].
75
Apoptosis is the main resistance mechanism against tumor development and it is fundamental for
76
cellular homeostasis [21]. B-cell lymphoma 2 (Bcl-2) is the primary member of the Bcl-2
77
family of regulatory proteins that regulate the molecular mechanisms of apoptosis by its dual
78
function of inhibiting or inducing apoptosis [22]. Members of this family include Bcl-2-
79
associated protein X (Bax) that induces and accelerates cell death when present as Bax/Bax
80
homodimer. Whereas, Bcl-2 and B-cell lymphoma extra-large (Bcl-xL) exert antiapoptotic action
81
when the heterodimers Bcl-2/Bax or Bcl-xL/Bax are formed [23, 24]. The interaction between
82
Bcl-2 family members determines the fate of the cell for death or survival [25].
83
The present study aimed at evaluating the antitumor activity of CPNPs-loaded DOX against
84
breast cancer MDA-MD-231.
85 86
2. Materials and Methods 2.1.Biological materials
87
MDA-MD-231 breast cancer cell line was purchased from the Holding Company for Biological
88
Product and Vaccines (VACSERA), Giza, Egypt. DOX and dimethyl sulfoxide (DMSO) were
89
purchased from Sigma-Aldrich (Germany).
90
2.2.Preparation of chitosan-protamine nanoparticles
91
The preparation of CPNPs was carried out according to Yue et al. [26], where 20 mg of chitosan
92
(Deacetylation degree 95%, molecular weight 80 kDa, Sigma Aldrich) was dissolved in 100 mL
93
of 0.1% acetic acid. pH 4.7 was adjusted using sodium hydroxide. Protamine (5 mg) was
94
dispersed in 4 mL ethanol, chitosan and protamine solutions were mixed for 30 min. by magnetic
95
stirring. 20 mg sodium tripolyphosphate (1mg/mL) was added drop by drop to the formed
96
mixture. The obtained NPs were centrifuged at 12,000 rpm for 15 min. and washed three times
97
with distilled water to ensure complete removal of non-encapsulated drug. The pellets were
98
freeze-dried and store at -4 oC till use.
99
2.3.Encapsulation of doxorubicin in chitosan-protamine nanoparticles
100
The preparation of DOX loaded on CPNPs were performed by the same method with the
101
addition of 3 mg DOX to the chitosan -protamine mixture before the dropping of
102
tripolyphosphatesodium (TPP).
103
2.4.Characterization of nanomaterial
104
CPNPs and DOX- CPNPs were suspended in phosphate buffer (pH = 7.4), and their sizes, zeta
105
potentials, and polydispersity index (PDI) were analyzed by Dynamic Light Scattering (DLS,
106
Zeta sizer Nano ZS, Malvern Instruments, Worcestershire, UK) at room temperature. The surface
107
morphology of NPs was observed by transmission electron microscopy (JEOL, JAM-2100-HR-
108
EM). Finally, the infrared spectra of free DOX, CPNPs and DOX- CPNPs were measured by a
109
Fourier-transform infrared (FT-IR) spectroscopy analyzer (Model JASCO FTIR-6100) within the
110
scanning range 4000–400 cm-1.
111 112
2.5. Determination of Encapsulation efficiency and drug loaded capacity
113
Five milligrams of freeze-dried DOX-CPNPs were vigorously vortexed in 1 mL methanol for 30
114
min. Then, the nanoparticles suspension was centrifuged at 12,000 rpm for 10 min. The
115
supernatant was used to determine the DOX content by measuring absorption at 480 nm by
116
(UV/VIS spectrophotometer, Shimadzu UV1800). Measurements were carried out three times,
117
then encapsulation efficiency (EE%) and loading capacity (LC %) were calculated by the
118
following equation: % = % =
119
ℎ
100
!
100
2.6.In vitro drug release study
120
Twenty milligrams of freeze-dried DOX-CPNPs were placed into a regenerated cellulose
121
dialysis bag (MWCO, 8000–14000, Sigma, St. Louis, MO). The closed bag was immersed into
122
50 mL of release medium (PBS pH = 5.0, 6.8, 7.4, 0.1 mol/L). The dialysis bag was incubated at
123
37oC under gentle agitation. At specific time intervals, 1 mL of release medium was removed for
124
DOX determination using spectrophotometric analysis at 480 nm. Each batch of experiments
125
was performed in triplicate. Cumulative percentage of DOX released was obtained by dividing
126
the cumulative amount of DOX released at each sampling time point (Mt) to the initial weight of
127
the CPNPs-DOX in the sample (M0), as presented in the following equation:
Cumulative release percentage = 1
Mt X 100 456 M0 4
128 129
2.7.Cell lines maintenance and drug treatment
130
The human triple-negative breast cancer cell line MDA-MB-231 was maintained on DMEM
131
media supplemented with antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin, Life
132
Technologies, NY, USA) and 10% FBS (Sigma-Aldrich). Cells were cultured in incubator at 37
133
˚C with 5% CO2 24 h prior treatment with three concentrations of free DOX concentration (1, 2,
134
and 3 µM in 0.05% DMSO), CPNPs or CPNPs-DOX (1mg/mL, 2 mg/mL, 5 mg/mL) at 70%
135
confluence for 48 h.
136
2.8.Trypan blue assay
137
Viable MDA-MB-231 cells were cultured in a 12-well tissue culture plate and treated with
138
different concentrations of free DOX, CPNPs, or CPNPs-DOX for 48 h. To assess the viability,
139
adherent cells were dislodged with 0.25% EDTA trypsin (Sigma-Aldrich). Cells were harvested
140
at low speed centrifugation and resuspended in 1 mL of normal growth medium. Equal volume
141
of cells and 0.4% trypan blue dye was mixed and left for 3 min at room temperature and then
142
loaded on hemocytometry slide. The viable and dead cells were counted under light microscope
143
(Olympus, BX43). Four readings were taken for each well, and the average was calculated. The
144
number of viable cells was calculated as follows:
145 146
Cell count = Average cell counts for the four readings x 2 x 104. 2.9.Cytotoxicity assay
147
Cell viability of MDA-MB-231 was determined by using 3-(4,5- dimethylthiazol-2-yl)-2,5-
148
diphenyl-tetrazolium bromide (MTT). Cells were seeded on 12-well plate with concentration of
149
5 x104 cells/well. Seeded wells were treated for 48 h with designated concentrations of DOX in
150
triplicates, and the rest of wells remained untreated to serve as control. After incubation, the
151
culture medium was replaced with 150 µL fresh media and 50 µL MTT (5 mg/mL in PBS), and
152
the plate was incubated for 4 h at 37 ˚C in a humidified atmosphere with 5% CO2. The developed
153
formazan crystals were dissolved by adding 200 µL of DMSO. The plate was incubated for 30
154
min. at 37 ˚C and then the optical density was determined at 550 nm using a spectrophotometric
155
microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).
156
2.10.
Cell cycle analysis
157
To assess the changes in the cell cycle after DOX treatment, 4 × 104 cells per well in a 12-wells
158
plate were seeded and treated with free DOX, CPNPs or CPNPs-DOX as previously described.
159
Cells were collected 48 h post-treatment at 600 rpm for 5 min. in cold 70% ethanol in PBS.
160
Cells, then, were centrifuged again at 600 rpm for 5 min. and left at 4 ºC for 2 h. Cells were
161
treated with 50 µg/mL PI, 0.1% Triton X-100 and 50 µg/mL RNAse for 25 min. and incubated at
162
room temperature in a dark place. The PI fluorescence was read on a FACScan flow cytometer
163
(BD FACSCalibur™). Data were analyzed to show the cell cycle distribution of the treated and
164
untreated cells.
165
2.11.
Apoptosis detection
166
MDA-MB-231 breast cancer cells were cultured in 12-well tissue culture plate and were treated
167
with free DOX, empty CPNPs, or CPNPs-DOX for 48 h. The apoptotic cells were identified
168
using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. Briefly,
169
treated and control cells were harvested and resuspended in 100 µL Annexin V binding buffer
170
and 5 µL Annexin V Alexa Fluor 488. The mixture was incubated for 15 min. in a dark place. PI
171
(4 µL) diluted in 1x Annexin V binding buffer (1: 10) was added and the mixture and incubated
172
again for 15 min. in a dark place.
173
Annexin V binding buffer (500 µL) was added to wash the Annexin/PI stained cells. Annexin/PI
174
staining was visualized on flow cytometer (BD FACSCalibur™). Annexin V-FITC binding was
175
analyzed at Ex = 488 nm; Em = 530 nm using FITC signal detector (FL1) and PI staining by the
176
phycoerythrin emission signal detector (FL2). 2.12.
177
RNA extraction and cDNA synthesis
178
Total RNA was extracted from control, free DOX-treated, empty CPNPs-treated, and CPNPs-
179
DOX-treated cells using RNA Isolation System (Qiagen, GmbH, Germany). RNA Quality was
180
evaluated by agarose gel electrophoresis and RNA concentration was measured by A260/A280
181
using NanoDrop 1000 Spectrophotometer (Wilmington, DE, USA). To synthesize cDNA, 5 µg
182
of the extracted RNA was mixed with 1µg random 6-mer primers and 1µM deoxyribonucleotides
183
(dNTP), 10 units of M-MLV SuperScript II Reverse Transcriptase (Invitrogen). The mixture was
184
incubated for 60 min. at 42 °C. 2.13.
185
Gene expression analysis
186
Quantitative real time PCR was used to amplify B-cl-2 gene. Primer sequences used in this study
187
are
188
CAATCCTCCCCCAGTTCACC-3'. Primers were generated using Primer-BLAST tool, NCBI.
189
About 100 ng (2 µL) of cDNA was mixed with 12.5 µL Cyber Green master mix, 10 pM (1.5
190
µL) of each of the primers, and the final volume was brought to 25 µL with molecular biology-
Bcl-2
forward:
5'-TCTCATGCCAAGGGGGAAAC-3'
and
Bcl-2
reverse:
5'-
191
grade water. The thermal profile was as follows: pre-PCR heating for 5 min. at 95 ºC, then 35
192
cycles of 94 ºC for 40 sec., 56 ºC for 45 sec., 72 ºC for 50 sec. followed by a final extension step
193
of 72 ºC for 10 min. All reactions were performed in triplicates on StepOne Plus thermal cycler
194
(Applied Biosystems, UK). 2-∆∆CT method was used to calculate the fold change in Bcl-2 gene
195
expression.
196
2.14.
Statistical analysis
197
Results of all experiments were presented as mean ± standard error (SE) in triplicate
198
experiments. All analyses were performed using the student's t-test to compare control with
199
treatment. P<0.05 was considered significant.
200 201
3. Results 3.1.Characterization of nanomaterial
202
As shown in (Fig. 1) the size distribution of CPNPs ranged from 90 to 800 nm with main peak at
203
163.50 nm and polydispersity index (PDI) of 0.512. While the CPNPs-DOX showed relatively
204
narrow particle size distribution ranged from 90 to 200 nm with main peak at 117 nm and PDI
205
value of 0.653. The zeta potential was 5.43 mV and 30.3 mV for CPNPs and CPNPs-DOX,
206
respectively (Fig. 1). The positive zeta potential was due to the residual amine groups. TEM
207
analysis revealed that the obtained nanoparticles were spherical and regular in shape and range in
208
size 100-150 nm (Fig. 2A). Finally, FTIR spectra of DOX, CPNPs, and CPNPs-DOX has several
209
bands as compared to those of free DOX or CPNPs. In brief, the FTIR spectrum of DOX shows
210
characteristic peaks at 3080 (C–H), 1730 (C=O) cm-1. These peaks are present in the FTIR
211
spectrum of CPNPs-DOX with small shift at 3050 (C–H), 1750 (C=O) cm-1. These results
212
indicate the successful loading of DOX in CPNPs (Fig. 2B).
213
3.2.Encapsulation efficiency and drug loading capacity
214
The encapsulation efficiency and drug loaded capacity were calculated according
215
aforementioned equations (materials and Methods section). Data obtained showed encapsulation
216
efficiency of 21% and the drug loaded capacity of 3.65%. According to these results, we
217
determined the concentrations of the CPNPs and CPNPs-DOX applied to the cells; 2.77 µg, 5.54
218
µg and 8.31µg, which contain the equivalent concentrations of the free DOX (1 µM, 2 µM and 3
219
µM).
220
Fig. 1
221
Fig. 2
222 223
3.3.In vitro release study
224
In the present study, we evaluated the release profile of DOX from CPNPs at three different pH
225
values. A quick release of DOX was seen in the first 6 h (approximately 40%, 28% and 16% of
226
the drug were released at pH 5.0, 6.8 and 7.4, respectively) then a relatively slow and sustained
227
release was observed in the following hours. In general, the amount of drug released at acidic
228
condition (pH 5.0) was greater than in other pH values (Fig. 3).
229
230
231
Fig. 3
232
3.4.Cytotoxicity assay
233
Cytotoxicity of the MDA-MB-231 breast cancer cells treated with free DOX, CPNPs, or CPNPs-
234
DOX was assessed by MTT assay and trypan blue dye exclusion test. Cells were incubated with
235
the drugs for 48 h and then harvested to assess viability. MTT assay results indicated that
236
CPNPs-DOX significantly reduced the overall cell viability compared to CPNPs and the free
237
DOX. The best CPNPs-DOX concentration was 3 µg/mL, where it resulted in 2% cell viability,
238
whereas free DOX at the same concentration resulted in 35% cell viability (Fig. 4).
239
Fig. 4
240 241 242
3.5.Cell cycle analysis
243
DNA content and apoptosis rate were analyzed in MDA-MB-231 cells treated with different
244
concentrations of encapsulated or free DOX. Flow cytometry data showed that CPNPs-DOX
245
(3µg/mL) yielded the highest percentage of cell growth arrest at G2/M stage compared to control
246
and free DOX (Fig. 5). Apoptosis was also detected in the treated and untreated cells using
247
PI/Annexin V-FITC staining. Data revealed that the percentages of cells undergone late
248
apoptosis were 1.63%, 14.26%, 11.11%, and 28.72% in control, free DOX treatment, CPNPs
249
treatment, and CPNPs-DOX treatment, respectively (Fig. 6).
250 251
Fig. 5
252
Fig. 6
253 254
3.6.Gene expression analysis
255
Bcl-2 is believed to be an apoptosis suppressor gene, and the upregulation of the protein in
256
cancer cells may interrupt the onset of apoptosis. In the present study Bcl-2 was downregulated
257
in breast cancer cells treated with different concentrations of free DOX, CPNPs, and CPNPs-
258
DOX. qPCR data showed that the level of Bcl-2 expression was negatively correlated with
259
elevation of CPNPs-DOX concentration, where it reached 0.04% in the highest concentration (3
260
µg/mL) relative to untreated cells and house-keeping gene (Fig. 7).
261
Fig. 7
262 263 264 265
4. Discussion 4.1.In vitro release study
266
Naturally, pH is gradient in the endosomal or lysosomal of tumor cells (5.0–6.5) and tumor
267
microenvironment (6.5–7.2) [27]. pH-sensitive nanoparticles might actively release drug at the
268
tumor site or in the endosomal or lysosomal of target tumor cells, which can enhance the
269
antitumor activity, as well as reduce potential damage to normal cells.
270
The significant decrease in the count of viable cells exhibited by CPNPs-DOX treatment might
271
indicate that the carrier CPNPs enhanced the bioavailability of DOX. Interestingly, the empty
272
CPNPs showed no toxicity in concentrations up to 5 mg/mL, which proved the biocompatible
273
nature of this nanocarrier. Taking these two findings together, we can conclude that the severe
274
decrease in cell viability is attributed mainly to DOX released from CPNPs. Other studies
275
indicated the obtained profile although in other types of cancers. Fang et al., [28] reported a
276
significantly higher intracellular DOX concentrations and more apoptotic induction in lymphoma
277
cells treated with DOX-loaded nanocarrier. DOX-loaded CNTs can be released in a sustained
278
manner and exert an effective antitumor activity in cervical cancer cells, making it potentially
279
promising therapeutic option in cancer treatment [29]. Our in vitro release experiment revealed
280
that releasing DOX from CPNPs-DOX was pH-dependent, where 40%, 28% and 16% of the
281
drug were released at pH values of 5.0, 6.8 and 7.4, respectively. When DOX was loaded on
282
MgO nanoflakes, it exhibited releasing rates of 90.2%, 50.5%, and 10% at pH values of 3, 5.0,
283
and 7.2, respectively [30]. These data might indicate the superiority of MgO nanoflakes as a
284
nanocarrier over CPNPs. Das et al. [31] indicated that DOX-loaded nanoceria exhibited higher
285
cellular uptake and drug release rates compared with free DOX, and this release is enhanced
286
under reductive acidic conditions (pH 5.0, 10mM glutathione) than under physiological
287
conditions (pH 7.4). Besides its enhanced bioavailability, DOX-loaded nanocarriers (such as
288
DOX-Fe-PDA/FA-PEG) can also trigger intracellular ROS overproduction, thereby enhancing
289
its therapeutic effect on breast cancer [32]. Other approaches use chitosan nanobubbles (NBs) as
290
a carrier for delivering DOX in cancer cells. Zhou et al. [33] reported the efficiency of DOX-NBs
291
to release DOX in MCF-7 breast cancer cells.
292
4.2.Encapsulated DOX and cell viability
293
Generally, the size and surface properties of NPs have a vital role in drug release, in vivo
294
pharmacokinetics, and cellular uptake [34]. The tiny size allows nanoparticles to circulate
295
through the microvascular bed of a tumor and extravasate into the perivascular space by
296
convective transport through the endothelium and retained at the site [35]. In the present study,
297
the obtained DOX- CPNPs had a nano size and relatively small PDI value that indicated that the
298
size distribution of DOX-NPs was narrow.
299
Breast cancer cells were treated with different doses of free DOX, CPNPs or CPNPs-DOX for 48
300
h to elucidate the efficiency of encapsulated DOX in inducing apoptosis via downregulating Bcl-
301
2. MDA-MB-231 breast cancer cell viability assays revealed a very significant decrease (p =
302
0.00026) in the percentage of viable cells treated with CPNPs-DOX (2%) at the highest
303
concentration (3 µg/mL) compared to free DOX (p = 0.002) and empty CPNPs (p = 0.08). It has
304
been reported that DOX-loaded PLA-TPGS nanoparticles (with about 100 nm in size) also
305
exhibited higher cytotoxicity and cellular uptake on both HeLa and HT29 cells [36]. Meanwhile,
306
other composites such as DOX-CeO2 also showed higher cell proliferation inhibition in ovarian
307
cancer compared with free DOX. A recent study indicated that the free DOX resulted in 35% and
308
89% cytotoxicity when MCF-7 cells treated with 10 µM and 2500 µM, respectively [37].
309
Although its benefits, using DOX in free form requires higher doses, which can be harmful to the
310
normal cells. Thus, loading DOX to a nanocarrier represents a safe way to treat cancer.
311
4.3.Apoptosis induction
312
Programmed cell death occurs as a cellular response to either external or internal stimuli. Thus,
313
regulation of this process is crucial for normal growth and homeostasis. Inducing apoptosis,
314
especially via disrupting the apoptotic machinery, is a proved way to treat cancer. In the present
315
study, we treated breast cancer cells with different concentrations of free DOX and DOX-loaded
316
CPNPs for 48 h. Results indicated that DOX-loaded CPNPs treatment (eq. to 3 µg/mL) has
317
resulted in a significant increase (p = 0.049) in the percentage of apoptosis (28.72%) compared
318
with control (1.63%), making this composite a potential treatment for breast cancer. Several
319
studies indicated the efficiency of loading DOX on nanocarrier in apoptosis induction. Siddharth
320
et al. [38] reported that DOX-loaded-PLGA-PVA-NP enhanced the uptake of DOX in MCF-7-
321
DOX-R cells and caused apoptosis by increasing both apoptotic nuclei and Bax/Bcl-xL ratio.
322
DOX-loaded FA-Se induced apoptosis rates (24.77%) in cervical cancer cells compared with the
323
cells treated with free DOX (10.48%). These data indicated that DOX-loaded FA-Se could
324
augment the antitumor activity of DOX in cervical cancer [39]. Furthermore, DOX-loaded CNPs
325
conjugated with FA caused enhanced release of cytochrome c as well as the activation of
326
downstream caspases to assist apoptosis in Y-79 retinoblastoma cells, which may provide a drug
327
delivery system (e.g. DOX) for the treatment of retinoblastoma [40]. In conclusion, nanocarriers-
328
mediated drug delivery offers a safe, fast, and reliable approach to augment the available
329
strategies to combat cancer.
330
4.4.Cell cycle analysis
331
In the present investigation, we analyzed the breast cancer cells after being treated with DOX or
332
CPNPs-DOX composite using PI staining. Data obtained indicated that CPNPs-DOX treatment
333
has resulted in the highest percentage of pre-G1 (28.72%) compared with control (1.53%), free
334
DOX (14.26%), and CPNPs (11.11%), which represents the effect of this composite in triggering
335
cell death. Other composites such as DOX-loaded FA-Se showed greater activity to induce
336
apoptosis in cervical cancer cells compared with free DOX. Pre-G1 apoptosis peak was 29.58%
337
and 13.69% when cells were treated with DOX-loaded FA-Se and free DOX, respectively [39].
338
Furthermore, treating MCF-7 breast cancer cells with DOX- loaded- PLGA-PVA-NPs has
339
resulted in a notable increase of Sub-G1, indicating the efficiency of this composite in inducing
340
apoptosis [38]. Meanwhile, our prepared composite arrested breast cancer cells at G2/M phase
341
(47.18%), which indicate its antiproliferative effect, and hence its efficacy as a therapeutic agent
342
for cancer. Neither DOX nor CPNPs did not exhibit this effect. MCF-7 treated with DOX-loaded
343
MagAlg (at 5 µg/mL and 0.5 µg/mL) showed an increase in the number of cells in G2/M phase
344
(20%) [41]. This might indicate that CPNPs-DOX is more efficient than DOX-loaded MagAlg in
345
inhibiting breast cancer proliferation.
346
4.5.Gene expression analysis
347
We analyzed the expression of Bcl-2 gene in treated and untreated cells. Results indicated that
348
DOX-loaded CPNPs treatment has resulted in a significant decrease in Bcl-2 expression (87%)
349
compared with CNPNs (72%) and free DOX (84%). Bcl-2 serves as an inhibitor of the intrinsic
350
(mitochondrial) apoptosis pathway, where it blocks Bid and Bax to prevent the release of
351
cytochrome c, that activates caspase cascade. Thus, downregulation of Bcl-2 is the main
352
triggering factor for the cells to undergo apoptosis [42]. It was indicated that DOX decreases the
353
anti-apoptotic Bcl-xL and increases pro-apoptotic Bax mRNA levels [43], leading to the
354
execution of apoptosis. Increasing the bioavailability of DOX within tumor might help
355
completely remove these cells. Other studies indicated the downregulation of Bcl-2 in different
356
cells including MDA-MB-231 breast cancer cells, MTLn3 adenocarcinoma cells treated with
357
DOX [44-46].
358
5. Conclusion
359
The main drawback of chemotherapy is the decreased bioavailability within cancer cells. In the
360
present investigation, novel protamine-chitosan pH-sensitive DOX nanoparticles were developed
361
and characterized using different tools, including TEM, FT-IR, zeta potential and size. We
362
treated MDA-MB-231 breast cancer cells with different concentrations of this composite along
363
with free DOX and empty carrier. Our data indicated that treating cells has resulted in a
364
significant decrease in the cell viability and cell count. Furthermore, apoptosis rate and cell cycle
365
phase distributions were also analyzed. All obtained data showed the efficacy of the DOX-loaded
366
CPNPs in inducing apoptosis and arresting cells at G2/M phase, indicting the antiproliferative
367
activity of the composite compared with free DOX and the empty carrier. This study concludes
368
that DOX-loaded CPNPs represents an efficient combination to enhance the chemotherapeutic
369
action of DOX in treating breast cancer.
370 371
Author participation
372
MA: conceptualized the idea, OM and SEA: conducted the experimental work, EC: prepared
373
drawings and illustrations, HS: wrote the manuscript.
374
Conflict of interest
375
The authors declare no conflict of interests
376
Acknowledgement
377
This work received no fund from any funding bodies
378 379
References
380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396
Figures and Ligands
397
Fig. 1
398 399 400 401 402 403 404 405
Fig. 1: Analysis of composite zeta size and zeta potential. A: CPNPs zeta size, B: CPNPs-DOX
406
zeta size, C: CPNPs zeta potential, and D: CPNPs-DOX zeta potential.
407
408
409
Fig. 2
A 410 411 412 413 414 415
B
416
Fig. 2: A: TEM image of CPNPs-DOX reveals the spherical shape of the composite that range in
417
size 100-150 nm. B: FTIR spectroscopic analysis reveals three distinct chemical composition of
418
a: CPNPs, b: free DOX, and c: CPNPs-DOX.
419 420 421 422 423 424 425
426
427 428 429 430 431 432 433
Fig. 3
434
435
436
Fig. 3: In vitro release profile of DOX from CPNPs at three different pH values. A quick release
437
of DOX was seen in the first 6 h with approximately 40%, 28% and 16% of the drug was
438
released at pH 5.0, 6.8 and 7.4 respectively. In general, the amount of drug released at acidic
439
condition (pH 5.0) was greater than other pH values.
440 441 442 443 444 445
446
Fig. 4
447 A 448 449 450 451 452
453
B
454 455 456 457 458 459 460 461 462
Fig. 4: Cell viability and cell count. A: MTT assay revealed a significant decrease in the breast
463
cancer cell viability after being treated with CPNPs-DOX compared with free DOX and CPNPs.
464
B: Cell count using trypan blue indicated a significant decrease in the count of cells treated with
465
the prepared composite compared with free DOX and CPNPs.
466
467
Fig. 5
468 469 470 471 472 473 474 475 476 477 478 479 480 481 482
Fig. 5: Cell cycle distribution and DNA content. The distribution of cell cycle phases after
483
treating breast cancer cells with DOX, CPNPs, and DOX-loaded CPNPs. Data revealed an arrest
484
at G2/M phase when cells treated with the prepared composite CPNPs-DOX for 48 h at 3 µM.
485
486
Fig. 6 487 488 489
490 491 492 493 494 495 496
Fig. 6: Apoptosis detection. MDA-MB-231 breast cancer cells were treated with DOX, CPNPs,
497
and DOX-loaded CPNPs for 48 h at 3 µM. Annexin-V FT-IC/PI staining indicated a significant
498
increase in the apoptosis rate in cells treated with DOX-loaded CPNPs compared with those
499
treated with frees DOX or CPNPs.
500
501
Fig. 7
502 503 504 505 506 507
508 509 510 511 512 513 514 515 516
Fig. 7: Gene expression analysis of Bcl-2. Treated and untreated cells were subjected to real time
517
PCR to assess the changes in Bcl-2 gene expression as a result of different treatment. Data
518
indicated that cells treated with DOX-loaded CPNPs showed a significant reduction in the
519
expression of Bcl-2 compared with cells treated with either free DOX or empty carrier.
520
521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565
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Conflict of interest All the authors of: “Doxorubicin Loaded on Chitosan-protamine Nanoparticles Triggers Apoptosis via Downregulating Bcl-2 in Breast Cancer Cells” Mohamed Abdel-Hakeem, Omnia Magdy, Shaimaa E. Abdel-Ghany, Emre Cevik and Hussein Sabit3 declare no conflict of interests.