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Improving plasma stability and antitumor effect of gemcitabine via PEGylated liposome prepared by active drug loading
Journal Pre-proof Improving plasma stability and antitumor effect of gemcitabine via PEGylated liposome prepared by active drug loading Ning Ding, Yaxi Wang, Wei Chu, Tian Yin, Jingxin Gou, Haibing He, Yu Zhang, Xing Tang, Xiaolin Wang, Yanjiao Wang PII:
S1773-2247(19)31244-4
DOI:
https://doi.org/10.1016/j.jddst.2020.101538
Reference:
JDDST 101538
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 22 August 2019 Revised Date:
11 January 2020
Accepted Date: 22 January 2020
Please cite this article as: N. Ding, Y. Wang, W. Chu, T. Yin, J. Gou, H. He, Y. Zhang, X. Tang, X. Wang, Y. Wang, Improving plasma stability and antitumor effect of gemcitabine via PEGylated liposome prepared by active drug loading, Journal of Drug Delivery Science and Technology (2020), doi: https:// doi.org/10.1016/j.jddst.2020.101538. 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. © 2020 Published by Elsevier B.V.
Author Statement Ning Ding: Conceptualization, Methodology, Software, Writing - Original Draft, Writing - Review & Editing Yaxi Wang: Conceptualization, Methodology, Writing - Original Draft Xiaolin Wang: Methodology Wei Chu: Resources Tian Yin: Validation Jingxin Gou: Investigation Haibing He *: Supervision，Visualization，Yanjiao Wang Yu Zhang： ：Validation Yanjiao Wang： ：Investigation ：Project administration，Funding acquisition Xing Tang：
Improving plasma stability and antitumor effect of gemcitabine via PEGylated liposome prepared by active drug loading
Ning Dinga, Yaxi Wanga, Xiaolin Wanga, Wei Chua, Tian Yinb, Jingxin Goua, Haibing Hea *, Yu Zhanga ,Yanjiao Wanga, Xing Tanga
a
Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical
University, Shenyang 110016, Liaoning, PR China b
School of Functional Food and Wine, Shenyang Pharmaceutical University,
Shenyang 110016, Liaoning, PR China
Corresponding author: Name: Haibing He Email: hhb_emily @126.com Telephone: +86 02443520558 Fax numbers: +86 02423911736
Present address： Shenyang Pharmaceutical University, Wenhua Road 103 Shenyang, 110016 Liaoning Province, People’s Republic of China
Graphical abstract
1
Improving plasma stability and antitumor effect of gemcitabine via
2
PEGylated liposome prepared by active drug loading
3 4
Ning Dinga, Yaxi Wanga, Wei Chua, Tian Yinb, Jingxin Goua, Haibing Hea *, Yu
5
Zhanga , Xing Tanga
6 7 8 9 10
a Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, Liaoning, PR China b School of Functional Food and Wine, Shenyang Pharmaceutical University, Shenyang 110016, Liaoning, PR China
11 12
Corresponding author:
13
Name: Haibing He
14
Email: hhb_emily @126.com
15
Telephone: +86 02443520558
16
Fax numbers: +86 02423911736
17 18
Abstract
19
Gemcitabine, as a nucleoside analog, has been used as first-line chemotherapy
20
drug for many years. However, the treatment dose of gemcitabine in the clinic has
21
been usually extremely high due to its rapid metabolism. The aim of this study was to
22
achieve successful chemotherapy at low doses by a PEGylated liposomal delivery
23
system. The liposomes (GEM-Lip) were prepared with Lipoid S-100, Cholesterol and
24
DSPE-PEG2000
25
dispersion-extrusion-ammonium sulfate gradient method. The entrapment efficiency
26
(EE, %) and drug loading (DL, %) of the liposomes (117.8 ± 16.85 nm) were 68.83 %
27
and 2.52 %, respectively. The in vitro release study and plasma stability experiments
28
showed the GEM-Lip was more stable than GEM-Sol at pH 7.4 and in the plasma.
29
AUC and Cmax of GEM-Lip were shown to be 6.20 and 16.44-fold higher than that of
(at
a
molar
ratio
of
9:2:0.07)
by
the
film
30
the solutions.30 The pharmacodynamics experiment in Kunming mice bearing a H22
31
cancer cell model showed that the tumor inhibition rate of gemcitabine liposomes was
32
6.25-fold as that of gemcitabine solutions when administrated intravenously. Our
33
findings demonstrated that the preparation of GEM-Lip could effectively enhance the
34
pharmacokinetic properties, the plasma stability and antitumor effect of gemcitabine.
35 36
Key Words：Antitumor effect
Gemcitabine
Liposome Plasma stability
37 38
Scheme 1. Active drug loading mechanism of GEM-Lip and its therapeutic efficacy.
39 40
1.Introduction
41
Gemcitabine (2c, 2c-difluoro-2c-deoxycytidine, GEM) is a deoxycytidine
42
antimetabolite, and has shown excellent efficacy both alone and in combination for
43
the treatment of various malignancies, including ovarian cancer, pancreatic cancer,
44
non-small cell lung cancer as well as other solid tumors [1-3]. The anti-tumor
45
mechanism of gemcitabine is to be absorbed as a prodrug by cells and phosphorylated
46
within the cell to form the active portion of gemcitabine triphosphate, which inhibits
47
DNA synthesis [4, 5]. However, after systemic administration, gemcitabine was
48
rapidly converted into an inactive metabolite by cytidine deaminase and excreted
49
through the urine, which limits its antitumor effect and application in the clinic [6-8].
50
In order to reach a therapeutic level, gemcitabine is currently administered at a high
51
dose of 1,000 mg/m2 for 30 min intravenous (i.v.) infusion, which causes
52
hematotoxicity and other side effects. Therefore, there is a strong need to obtain a
53
preparation of gemcitabine with good plasma stability.
54
In the present studies, some prodrugs of gemcitabine with lipophilic acyl chains
55
were designed to overcome above problems. A lipophilic prodrug can be obtained by
56
linking the 4-amino group of gemcitabine with an acyl chain such as pentanoyl group,
57
heptanoyl group, lauroyl group, stearoyl group or squalenoyl derivative [9]. Studies
58
have shown that prodrugs could enhance the pharmacological activity of the active
59
compound compared to the drug administered in free form [9]. However, further
60
researches, such as safety studies, are still required to prove that there are few side
61
effects of prodrugs. Based on this, encapsulation of gemcitabine into liposomes could
62
be more readily applied into the clinic compared to prodrugs. Encapsulating within
63
liposomes may be a means to protect drugs from metabolic inactivation and reduce
64
the accumulation of drugs in healthy tissues which consequently alleviates the toxicity
65
and adverse effects [10, 11].However, liposomes were usually identificated by the
66
reticuloendothelial system and accumulated less to the target if there is no surface
67
modification which might limit the clinical trial function of gemcitabine. To overcome
68
this type of problem, PEGylated liposomes have been used as carriers for many drugs
69
to achieve a longer cycle time in vivo and have shown great promise for cancer
70
therapy applications [11, 12], notably as Doxil ®. According to the literature, the
71
PEGylated liposomal entrapment of drugs is able to produce a prolonged blood
72
circulation time and facilitate tumor accumulation, therefore effectively promoting the
73
pharmacokinetics (PK) and pharmacodynamics (PD) of the drug and ultimately
74
enhancing the antitumor activity [8]. In addition, studies have shown that GEM
75
liposomes have a significant sustained release in vitro compared with conventional
76
GEM injection [13]. Namely, liposomes could provide protection against rapid
77
metabolic inactivation of drugs, and thereby generate a greater anti-tumor effect in
78
vivo and improve the plasma stability of gemcitabine.
79
Several methods for preparing GEM liposomes (GEM-Lip) have been reported
80
in the literature [14-16], and can be mainly divided into two categories: passive drug
81
loading method (e.g. membrane hydration and reverse phase evaporation) and the
82
active loading method using the ammonium sulfate gradient. When encapsulating a
83
hydrophilic drug such as gemcitabine, the entrapment efficiency (EE%) of the
84
liposome fabricated with passive loading, using the membrane dispersion method and
85
the reverse evaporation method was about 47% [15] and 67% [14], respectively, but
86
the encapsulation conditions were not readily controlled. As well, the particle sizes of
87
the liposomes obtained from these two methods were over 1 µm, which makes it easy
88
to be eliminated by the reticuloendothelial system. However, when instead applying
89
the film dispersion-extrusion-ammonium sulfate gradient method, the liposome
90
particle size could be smaller and more uniform by extrusion and the EE of liposome
91
was slightly higher than above two methods by utilizing the ionic gradient between
92
the inner and outer sides of the phospholipid to form the medicine carrying motive
93
force.
94
According to previous literature [16], the ammonium sulfate gradient method,
95
which is one of the most widely used active loading methods for achieving preferable
96
liposomes, is suitable for loading weak alkaline drugs with pKa ≤ 11 and logP values
97
in the range of -2.5 to 2.0. Therefore, in our study, with a pKa and logP of 3.6 and -1.4,
98
respectively,
99
dispersion-extrusion-ammonium sulfate gradient method to prepare liposome. In
100
addition, by using Lipoid S-100 with little amount of DSPE-PEG2000, this liposome
101
seemed to be more economical than what other literatures reported. So this liposome
102
might be a good choice for gemcitabine to improve its stability in the systemic
103
circulation and antitumor effect.
gemcitabine
might
be
a
suitable
drug
for
the
membrane
104 105
2. Materials and Methods
106
2.1 Materials and Reagents
107
Lipoid E-80 (82% Phosphatidylcholine (PC), 9.2% Phosphatidyl ethanolamine
108
(PE)) and Lipoid S-100 (96% PC, < 0.1% PE) were purchased from Lipoid KG
109
(Ludwigshafen, Germany). PL-100M (78% PC, 18% PE), PC-98T (98.6% PC,< 0.1%
110
PE),and1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy(polyethylene
111
vglycol)-2000]
112
Technology Pharmaceutical Co., Ltd. (Shanghai, China). Cholesterol (CHO) was
(DSPE-MPEG2000) were obtained
from
Advanced
Vehicle
113
provided by J & K (China) and ammonium sulfate was purchased from Shanghai
114
Aladdin Biochemical Technology Co., Ltd (China). Sodium chloride was purchased
115
from Shandong Yuwang Group (China). GEM (HPLC purity >99%) was a kind gift
116
from Shenyang Jin Chang Pharmaceutical Co., Ltd (China). Chloroform, methanol,
117
ethylacetate and isopropanol were provided by Tianjin Concord Technology Co., Ltd.
118
(China). Formic acid was purchased from Thermo Fisher Scientific Inc. (America).
119
All other chemicals and reagents were of analytical or chromatographic grade.
120 121
2.2 Preparation of Gemcitabine-loaded liposomes (GEM-Lip)
122
GEM-Lip was prepared by the thin film dispersion-extrusion-ammonium sulfate
123
gradient method [17, 18]. First, the phospholipids, CHO and DSPE-MPEG2000 were
124
dissolved in a round-bottom flask using chloroform-methanol (3:1 v/v) solution at a
125
molar ratio of 9:2:0.07. The organic solvents were next removed by means of vacuum
126
rotatory evaporator at 40 °C. The resulting lipid film was further dried under a
127
nitrogen stream for 1 h, followed by hydrating and vortexing in 250 mM ammonium
128
sulphate to obtain a suspension of multilamellar vesicles [19]. The suspension was
129
then extruded (LiposoFast LF1, Avestin, Canada) through 100 nm polycarbonate
130
filters (Nucleopore) for 30 times and 50 nm polycarbonate filters for 20 times. To
131
remove the unencapsulated aqueous ammonium sulfate, the products were dialyzed in
132
a 150 mM sodium chloride solution at 35 °C for 6 h. gemcitabine at a concentration of
133
1 mg/mL was then incubated with the blank liposomes at 60 °C for 2 h to obtain the
134
GEM-Lip composite.
135 136
2.3 Characterization of GEM-Lip
137
2.3.1 Particle size analysis
138
The mean particle size and the polydispersity index (PI) of the GEM-Lip was
139
measured by photo correlation spectroscopy (PSC, dynamic light scattering, DLS)
140
with a Nicomp™ 380 particle sizing system (Santa Barbara, USA). Each sample was
141
recorded three times using ten sub-run measurements. To avoid multiple scattering
142
effects, a maximum of 200–300 HZ was adjusted by diluting of the samples with
143
purified water.
144
The morphology of the liposomes was examined by transmission electron
145
microscopy (TEM) (JEM-2100) using negative staining. Briefly, one drop of liposome
146
suspension was put on the top of a 3 mm 200-mesh copper grid. After 2 min
147
incubation, the surplus was removed by filter paper, and the liposome sample on the
148
mesh was stained with 2% phosphotungstic acid solution and incubated for another 2
149
min. The sample was dried at room temperature before use.
150 151
2.3.2 Entrapment Efficiency（ （EE） ）and Drug Loading (DL)
152
The EE of GEM-Lip was determined by centrifugal ultrafiltration. Briefly,
153
200µL GEM-Lip was demulsificated with 1 mL isopropyl alcohol, and diluted with
154
methanol to 10 mL. The total amount of GEM was acquired after ultracentrifugation
155
at 12,000 rpm for 10 minutes and quantified by HPLC in triplicate after filtering
156
through 0.22 µm microporous membranes. Liposomes (1.0 mL) were added to an
157
ultrafilter (Amicon ultra, Millipore Co., USA, MWCO 10 kDa) and centrifuged at
158
3,000 rpm for 15 min. In the ultrafiltrate, unencapsulated drug was obtained and
159
diluted to 10 mL with the mobile phase and quantified as above. HPLC conditions
160
were as follows: a C18 column (250 × 4.6 mm, 5 µm; Thermo Hypersil GOLD aQ,
161
America) was used, the mobile phase was a mixture of 20 mM sodium acetate
162
aqueous-methanol (90:10 [v/v]) at a flow rate of 1 mL/min, the column temperature
163
and wavelength were 25 °C and 268 nm, and the injection volume was 20 µL.
164
EE and DL were calculated using the following equations:
165
EE (%) = (1- WFree / WToal)×100%
166
DL (%) = Wen / WLip×100%
167
where WTotal was the total amount of drug of the preparation and WFree was the
168
amount of drug in ultrafiltrate, respectively. Wen was the amount of drug of entrapped
169
in the liposome, and WLip was the total amount of drug and excipients.
170 171 172
2.4 In vitro drug release study In vitro release of gemcitabine from GEM-Lip was investigated by the dynamic
173
dialysis method. 1 mL of GEM-Lip (n=3) or GEM-Sol (n=3) were placed into a
174
dialysis membrane (molecular weight cut-off 14 kDa, Spectrum Labs, Rancho
175
Dominguez, CA, USA) and incubated in 10 mL of release medium (isotonic pH 7.4
176
PBS, in order to simulate sink conditions in vivo) at 100 rpm under 37 °C with a
177
shaking
178
Manufacturing Co., Ltd, China). At predetermined time intervals (at 0.25, 0.5, 1, 2, 4,
179
6, 8, 12, and 24 h), the medium was withdrawn completely and replaced with 10 mL
180
fresh medium. Samples were then filtered through 0.22 µm filter membrane and
181
analyzed by HPLC to estimate the amount of drug released.
bath
(ZWF-110X30,
Shanghai
Zhicheng
Analytical
Instruments
182 183
2.5 Plasma stability
184
Stability in rat plasma was evaluated by incubating GEM-Sol or GEM-Lip in
185
100 % rat blank plasma at a concentration of 1 mg/mL at 37 ℃ for 24 h. Thereafter
186
50 µL of samples were collected every 2 hours. 20 µL of cytarabine hydrochloride at a
187
concentration of 0.1 mg/mL as an internal standard and 50 µL methanol were added
188
into each sample. Next, 1 mL isopropanol and 2.5 mL ethyl acetate were added into
189
the above mixture and vortexed and centrifuged at 12,000 rpm for 10 min. 3 mL of
190
the supernatant was transferred and evaporated to dryness under a 40 ℃ air stream.
191
The residue was redissloved in 400 µL mobile phase and vortexed for 10 min. The
192
mixture was centrifuged at 12,000 rpm for 10 min and 10 µL of the supernatant was
193
transferred for the content analysis by HPLC. In addition, the plasma stability in terms
194
of particle size of GEM-Lip was also assessed.
195 196
2.6 Pharmacokinetic studies
197
Male SD rats were purchased from Laboratory Animal Center of Shenyang
198
Pharmaceutical University. The rats were randomly divided into two groups (six rats
199
in each group). Each rat was treated with 4 mg/kg of free gemcitabine (GEM-Sol
200
group) or GEM-Lip (GEM-Lip group) through tail vein injection. Blood samples (0.5
201
mL) were removed from the retro-orbital plexus at various times, and then centrifuged
202
at 6,000 rpm for 10 min in a refrigerated centrifuge (Anhui USTC Zonkia Scientific
203
Instruments Co., Ltd, China). The upper plasma samples were stored in a -20 °C
204
freezer until use.
205
For analysis, plasma samples were diluted by adding 1 mL isopropanol and 2.5
206
mL ethyl acetate [20]. After vortex-mixing the samples for 10 min (Lamivudine as
207
internal standard), the mixture was then centrifuged for 10 min at 12,000 rpm. Then 2
208
mL supernatant was transferred and evaporated to dryness under nitrogen stream
209
(40 ℃). The sample residue was redissolved in 400 µL of mobile phase (methanol:
210
0.1% formic acid in water 80:20), and vortexed for 10 min, and then centrifuged at
211
12,000 rpm to obtain the supernatant for analysis by LC-MS / MS. The LC-MS/MS
212
conditions were as follows: The ion source was an electrospray ion source (ESI
213
source positive ion scan). The ion source temperature was 150 °C and the desolvation
214
gas temperature was 400 °C. The desolvation gas flow rate and the cone blowback gas
215
flow rate were 550 L•h-1 and 50 L• h-1. N2 and Ar were used as a solvent removal gas
216
a collision gas, respectively. The scanning method was multiple reactions monitoring
217
(MRM), and the detected m/z values of gemcitabine and lamivudine were
218
264.03/111.92 and 229.92/111.91, respectively. The capillary voltage was 3 kV. When
219
detecting gemcitabine, the cone voltage and collision energy were 20 V and 25 V,
220
respectively. And when the internal standard was detected, the cone voltage and
221
collision energy were 12 V and 10 V.
222 223
2.7 Antitumor activity of GEM-Lip in xenograft animals
224
Animal experiments were carried out in agreement with the principles and
225
procedures outlined by the local Ethical Committee. Mouse liver cancer cells H22 in
226
the exponential growth phase were collected and diluted with sterile saline to adjust
227
the concentration to 1×106 cells/mL. 0.1 mL of the above cell suspension was injected
228
into the peritoneal cavity of Kunming mice. Ascites were extracted under sterile
229
conditions from 7 to 10 days, and diluted to 1×106 cells/mL with sterile saline. The
230
mouse ascites (0.1 mL) were subcutaneously injected in the right flank regions of
231
mice to establish a tumor-bearing mouse model and allowed to grow for 1 week. The
232
Kunming mice were randomly distributed into 3 groups (n=10): saline (Control
233
group), free GEM (GEM-Sol group, 4 mg/kg) or GEM-Lip (GEM-Lip group, 4
234
mg/kg). On the seventh day after the establishment of the mouse tumor model, a
235
considerable dose of preparations or solution was injected into the tail vein every
236
other day for four times, and the control group was injected with 0.2 mL of saline.
237
The tumor volume and body weight of each mouse was measured every day. Tumor
238
volumes were calculated and plotted as average values per group. The sizes of tumor
239
masses were measured with a caliper and tumor volume was calculated according to
240
the formula (Paolino et al. 2010): V = 0.5 × ab2
241 242
where a is the larger perpendicular diameter and b is the smaller perpendicular diameter of the tumor, respectively.
243
The feeding behavior, activity of the mice and motor activity of mice were
244
observed as indicators of general health. The mice were sacrificed by cervical
245
dislocation on the next day after the last administration, and then the tumors were
246
removed and weighed. The tumor inhibition rate (TIR) was calculated as follows: TIR = 1 −
Mean tumor weight of treated group × 100% Mean tumor weight of control group
247
After, the tumor masses were eradicated and rinsed in saline for analysis. They
248
were fixed in 4 % (w/v) buffered formaldehyde (pH = 7.4) at room temperature,
249
dehydrated in alcohol and then embedded in paraffin. Sections with a thickness of 7–
250
10 µm were sliced using a microtome and stained following the eosin B / hematoxylin
251
method [21] (HE staining). The sections were then subjected to histopathological
252
examination under a microscope.
253 254
2.8 Statistical analysis
255
All experiments were performed at least three times and expressed as means ±
256
SD and results were dealed with Microsoft Excel 2010 and Graphpad prism 6. Data
257
were analyzed for statistical significance using Student’s test. p < 0.05 was considered
258
statistically significant, and p < 0.01 was considered highly significant.
259 260
3. Results
261
3.1 Characterization of gemcitabine loaded liposomes (GEM-Lip)
262
The EE of the liposomes was approximately 68.83 % with a final DL of
263
approximately 2.52 %. The particle size was around 117.8 ± 16.85 nm with a PDI of
264
0.020. The morphology of the particles was characterized by TEM in Fig.1.
265 266
Fig.1.TEM images of GEM-Lip (A and B), the particle size distribution of GEM-Lip
267
(C) and the structure of GEM-Lip (D).
268 269
3.2 In vitro release studies
270
A drug release study was performed to evaluate the drug release pattern and form
271
of drug release. The drug release was represented graphically by plotting percent drug
272
release against time, and the results are depicted in Fig.2. The cumulative release
273
percentage of the liposomes was 12.48 % at 1 h, whereas a quick diffusion of
274
GEM-Sol was observed at the same time point. By fitting the release curve to the
275
release model, it was found that the release of gemcitabine liposomes was in line with
276
the Ringer-Peppas model.
277 278
Fig.2. GEM-Lip and GEM-Sol were released in PBS at pH 7.4 (mean ± SD, n=3)
279 280
3.3 Plasma stability
281
As shown in Fig.3B, the percentage of drug remaining for GEM-Sol was about
282
20 % after incubating with rat plasma for 2 h, while that for GEM-Lip was above
283
90 %. The percentage of drug remaining was stable when incubating about 8 h, and
284
that for this two preparations were below 5 % (GEM-Sol) and above 50 % (GEM-Lip)
285
(p < 0.01) , respectively. And the particle size of GEM-Lip remained stable at 80 - 110
286
nm after incubating 8 h (p ＞ 0.05), which was consistent with the result of the
287
percentage of drug remaining.
288
Fig.3. The stability of particle size (A) and the percentage of drug remaining of
289
GEM-Lip (B) after incubating in the rat plasma for 24 h. Asterisks indicate significant
differences (t test; *p<0.05; **p<0.01)
290 291 292
3.4 Pharmacokinetics studies
293
In this experiment, the LC-MS / MS method was selected to determine the
294
plasma concentration of GEM-Lip and GEM-Sol. The mean gemcitabine plasma
295
concentration–time profiles after intravenous administration at a dose of 4 mg•kg-1 (n
296
= 6) of GEM-Sol and GEM-Lip are shown in Fig.4, and the important PK parameters
297
are listed in Table 1. As shown in Table 1, the AUC
298
group were 6.54 and 6.20-fold higher than that of the GEM-Sol group, respectively.
299
The t1/2z of GEM-Lip was around 2.89 h. The Vz and CLz of GEM-Lip were largely
300
decreased, and were 0.26 and 0.16-fold lower than that of the solutions. In addition,
301
the Cmax of GEM-Lip was nearly16.44-fold as that of GEM-Sol.
(0-t)
and AUC (0-∞) of GEM-Lip
302 303
Fig.4. Concentrations of GEM in male rat plasma after a single i.v. treatment (mean ±
304
SD, n = 6)
305 306
Table1. The Pharmacokinetics parameters of GEM-Sol and GEM-Lip Parameters
GEM-Sol
GEM-Lip
AUC(0-t)( ug/L*h)
2267.8 ± 273.47
14824.66 ± 467.60
AUC(0-∞)( ug/L*h)
2394.6± 353.67
14849.59 ±502.49
Cmax(ug/L)
608.9 ± 20.49
10010.3 ±1067.46
CLz(L/h/kg)
1.70 ± 0.22
0.27 ± 0.009
Vz(L/kg)
4.27 ± 0.73
1.11 ± 0.36
t1/2z(h)
1.77 ± 0.38
2.89 ± 1.04
307 308
3.5 Antitumor activity studies in mice
309
As shown in A and B of Fig.5, after 4 treatment doses, the volume of the tumors
310
in the mice treated with saline was 3329 mm3 and that with GEM-Sol was 2621 mm3,
311
while those of mice treated with GEM-Lip were smaller (∼1699 mm3). No evident
312
change in body weight was observed in the GEM-Lip group throughout the week of
313
study, however there was a slight drop in body weight for the control group compared
314
with other treatments. The results are expressed as an average, and T-tests were used
315
to determine statistical significance with p < 0.05 considered statistically significant
316
[22]. There were also differences in the survival status of mice in each administration
317
group. At the end of the administration period, four mice died in the control group due
318
to the deterioration of the condition and two died in the GEM-Sol group and
319
GEM-Lip group, and tumors of a mouse in this group were not obviously observed.
320
When comparing GEM-Lip and GEM-Sol, containing the same dose of gemcitabine,
321
the drug-loaded liposomes had an improved antitumor effect, and there was also a
322
significant difference compared with the normal saline group. The weight of the
323
collected tumor masses shown in Fig.5C confirmed these findings. Masses of ∼0.48 g
324
were observed in the case of GEM-Lip, while the weight of tumors in the control
325
group and the GEM-Sol group were ~1.23 g and ~1.11 g, respectively. Fig.5D
326
expresses the above differences. The tumor inhibition rate (TIR) of GEM-Sol was
327
9.76% and that of GEM-Lip was 60.98% (p < 0.05).
328
As shown in Fig.6, when the tumor slices were observed under microscope, a
329
large number of tumor cells were observed in the control group. In the GEM-Sol
330
group, tumor cells were deeply stained and nuclear pyknosis appeared, and necrotic
331
tumor cells were observed in the middle. In the GEM-Lip group, there were also
332
deeply stained, large, and multinucleated tumor cells, but the tumor cells in the
333
GEM-Lip group appeared to be relatively sparse comparing with the control group.
334
Fig.5. Tumor volume changes (A) , body weight changes (B) of various preparations
335
after i.v. injection in the tail, solid tumors stripped from H22-tumor-bearing mice(C)
336
and the tumor weight of different groups (D)
337 338
Fig.6. Histological analysis of mouse tumor excised of Control group (A), GEM-Sol
339
group (B), and GEM-Lip group (C) at the end of experiment
340 341
4. Discussion
342
As a major antitumor drug, gemcitabine has been widely applied in the treatment
343
of various tumors. However, it is easy to be metabolized by cytidine deaminase limits
344
its
antitumor
effect.
The
liposome
produced
by
the
film
345
dispersion-extrusion-ammonium sulfate gradient method might protect gemcitabine
346
from being metabolism. When GEM-Lip was prepared by film dispersion method, a
347
low EE (<30%) was obtained. And instability of particle size made it difficult to apply
348
reverse
349
dispersion-extrusion-ammonium sulfate gradient method might be superior to above
350
methods to a certain extent in our experiments. On one hand, particle size uniformity
351
(about 100 nm) could be guaranteed by extrusion. On the other hand, the role of
352
ammonium sulfate in the internal aqueous phase of the liposome was to provide an
353
acidic environment to elicit the protonation of gemcitabine, and thereby, distinctly
354
reduced the drug leakage from the liposome to a certain degree. In addition, due to the
355
difference of pH between the internal and external of liposome, there was a driving
356
force for unionized moiety to enter the internal of the liposome and be ionized.
357
According to the equilibration formula between the ionized and unionized
358
gemcitabine inside the liposome, the ratio of the amount of ionized and unionized part
359
was 2:1 when the equilibration was obtained [16]. So the max EE theoretically was
360
about 66.67%. Obviously, by applying the film dispersion-extrusion-ammonium
361
sulfate gradient method, the max EE was obtained in this experiment.
evaporation
method
to
the
final
preparation.
While
film
362
The in vitro release study was conducted at pH 7.4 to mimic the physiological
363
pH. The release profiles showed that gemcitabine in the liposomes could release more
364
slowly than in aqueous solution. A quick diffusion was observed in the GEM-Sol
365
group. However, the release profile of the liposomes was S-type, which suggested that
366
there was a fast release of GEM-Lip. And this might be due to the osmotic pressure
367
difference between the internal (550 mOsm) and external liposome (200 mOsm),
368
which caused it swelling with the influx of water [16]. In addition, the resistance of
369
the major release course mainly diffusion from the liposome inner water phase, so a
370
slow release phenomenon was observed next. Above results indicated that the
371
liposomes could be more stable than solutions at the physiological pH.
372
The plasma stability studies also confirmed above conclusion. The stability of
373
GEM-Lip was greater than GEM-Sol in rat plasma, which may be due to the
374
encapsulation of gemcitabine in the liposome and the protonation of gemcitabine
375
inside the liposome could reduce its exposure to plasma and prevent it from being
376
metabolized by cytidine deaminase, thus improving its stability in the rat plasma.
377
However, the particle size of GEM-Lip was decreasing with time and was stable from
378
8 h (p ＞ 0.05) which showed in the Fig 3A, and this indicated that the GEM-Lip
379
could maintain its structure when it existed in the blood.
380
The pharmacokinetics studies also verified the better stability of the liposomes.
381
As reported in literature, GEM-Sol was usually administered with larger doses in the
382
clinic to achieve therapeutic effects. This can cause many adverse effects such as
383
myelosuppression and impaired liver function. In this experiment, as stated above, the
384
plasma stability of GEM-Lip was confirmed to be better than GEM-Sol, thus the
385
preparation of GEM-Lip could be expected to reduce the required drug dose and
386
improve its antitumor effect. PEGylated liposomes could help gemcitabine circulating
387
for longer time in the systemic circulation and protected it from being metabolized. In
388
our experiment, gemcitabine was basically all encapsulated in the liposome after tail
389
vein injection, but GEM-Sol was unfortunate to meet the cytidine deaminase
390
immediately and was metabolized. So the Cmax of GEM-Lip was extremely higher
391
than GEM-Sol. However, as the time going on, GEM-Lip was constantly striking the
392
materials in the bloodstream while gradually cracked and gemcitabine in the inner
393
phase of liposome was exposed to the cytidine deaminase and was metabolized
394
rapidly, which caused the short t1/2. As well, other PK parameters of the liposome
395
which were compared with GEM-Sol confirmed that the encapsulation of GEM in
396
liposomes could limit the distribution range of drug in the systemic circulation[23].
397
For instance, the Vz and CLz of the liposomes were lower than the solutions, which
398
could reduce the possibility of active agent metabolism. Specifically, the lower Vz can
399
be an indication of reducing toxic side effects, and the lower CLz can represent the
400
long system cycle time and high stability of GEM-Lip. The t1/2z and Cmax of the
401
liposomes were longer and greatly larger respectively than the solutions, and this
402
indicated that the active agent could be stable in the blood for a longer time. All above
403
parameters confirmed that the GEM-Lip could protect gemcitabine from being
404
metabolized by cytidine deaminase.
405
The results of pharmacodynamics experiment showed the excellent antitumor
406
effect of the liposomes. The tumor volume and tumor weight of tumor-bearing mice
407
of the liposomes group were smaller than GEM-Sol group. The survival status of mice
408
in the liposomes group was also better than other groups. As Laquente studied, the
409
conventional dose of gemcitabine for mice was 100 mg/kg, and was administered on
410
days 0, 3, 6 and 9. And the TIR of the conventional schedule of gemcitabine was
411
about 97.59 % to human pancreatic carcinoma [24]. However, with so high treatment
412
dose, some side effects such as myelosuppression might be extremely harmful to
413
patients. Compared to this, though the TIR of GEM-Sol was extremely lower than
414
conventional dose, this lower treatment dose might cause fewer side effects and
415
relieve the suffering of patients. In addition, the TIR of GEM-Lip was 60.98 % even
416
with so low treatment dose.
417
These phenomenon might be because it always needs a high treatment dose for
418
gemcitabine to achieve the treatment effects. While the treatment dose of GEM-Sol
419
and GEM-Lip group in this study was 4 mg/kg, which was much lower than
420
conventional dose of gemcitabine. So the TIR of GEM-Sol was extremely lower than
421
expected. However, due to the protection of PEGylated phospholipids and the
422
encapsulation of gemcitabine, it was protected from cytidine deaminase metabolism
423
to a certain extent, thereby the body cycle time of gemcitabine was prolonged and the
424
AUC was increased. In addition, the presence of hydrophilic chain of
425
DSPE-MPEG2000 on the surfaces of liposomes was important for preventing their
426
uptake by the reticuloendothelial system and consequently for increasing the t1/2z of
427
liposomes and their prolonged presence in the bloodstream. In summary, the
428
antitumor activity of GEM-Lip was enhanced compared with GEM-Sol.
429 430
5. Conclusion
431
In this study, a GEM-Lip with a uniform particle size and high entrapment
432
efficiency was prepared by film dispersion-extrusion-ammonium sulfate gradient
433
method. The in vitro studies showed that the GEM-Lip could be more stable than
434
GEM-Sol at the physiological pH and in the rat plasma. The in vivo studies confirmed
435
that the GEM-Lip could protect gemcitabine from being metabolized by cytidine
436
deaminase. Furthermore, the pharmacodynamics results demonstrated that the
437
GEM-Lip could enhance antitumor activity effectively. In conclusion, the
438
encapsulation of gemcitabine in the liposome could improve its stability in the rat
439
plasma and enhance its antitumor effect to a certain degree.
440 441
Acknowledgments
442
We sincerely thank Amanda Pearce for the linguistic assistance during the
443
revision of this manuscript. There are no conflicts of interest within the authors. In
444
addition, the authors are very grateful to the 2016 Annual Youth Teachers’ Career
445
Development Support Program of Shenyang Pharmaceutical University [grant number
446
ZQN2016008].
447 448
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Declarations of interest: There was no conflict of interest among all authors.
S1773-2247(19)31244-4
DOI:
https://doi.org/10.1016/j.jddst.2020.101538
Reference:
JDDST 101538
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 22 August 2019 Revised Date:
11 January 2020
Accepted Date: 22 January 2020
Please cite this article as: N. Ding, Y. Wang, W. Chu, T. Yin, J. Gou, H. He, Y. Zhang, X. Tang, X. Wang, Y. Wang, Improving plasma stability and antitumor effect of gemcitabine via PEGylated liposome prepared by active drug loading, Journal of Drug Delivery Science and Technology (2020), doi: https:// doi.org/10.1016/j.jddst.2020.101538. 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. © 2020 Published by Elsevier B.V.
Author Statement Ning Ding: Conceptualization, Methodology, Software, Writing - Original Draft, Writing - Review & Editing Yaxi Wang: Conceptualization, Methodology, Writing - Original Draft Xiaolin Wang: Methodology Wei Chu: Resources Tian Yin: Validation Jingxin Gou: Investigation Haibing He *: Supervision，Visualization，Yanjiao Wang Yu Zhang： ：Validation Yanjiao Wang： ：Investigation ：Project administration，Funding acquisition Xing Tang：
Improving plasma stability and antitumor effect of gemcitabine via PEGylated liposome prepared by active drug loading
Ning Dinga, Yaxi Wanga, Xiaolin Wanga, Wei Chua, Tian Yinb, Jingxin Goua, Haibing Hea *, Yu Zhanga ,Yanjiao Wanga, Xing Tanga
a
Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical
University, Shenyang 110016, Liaoning, PR China b
School of Functional Food and Wine, Shenyang Pharmaceutical University,
Shenyang 110016, Liaoning, PR China
Corresponding author: Name: Haibing He Email: hhb_emily @126.com Telephone: +86 02443520558 Fax numbers: +86 02423911736
Present address： Shenyang Pharmaceutical University, Wenhua Road 103 Shenyang, 110016 Liaoning Province, People’s Republic of China
Graphical abstract
1
Improving plasma stability and antitumor effect of gemcitabine via
2
PEGylated liposome prepared by active drug loading
3 4
Ning Dinga, Yaxi Wanga, Wei Chua, Tian Yinb, Jingxin Goua, Haibing Hea *, Yu
5
Zhanga , Xing Tanga
6 7 8 9 10
a Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, Liaoning, PR China b School of Functional Food and Wine, Shenyang Pharmaceutical University, Shenyang 110016, Liaoning, PR China
11 12
Corresponding author:
13
Name: Haibing He
14
Email: hhb_emily @126.com
15
Telephone: +86 02443520558
16
Fax numbers: +86 02423911736
17 18
Abstract
19
Gemcitabine, as a nucleoside analog, has been used as first-line chemotherapy
20
drug for many years. However, the treatment dose of gemcitabine in the clinic has
21
been usually extremely high due to its rapid metabolism. The aim of this study was to
22
achieve successful chemotherapy at low doses by a PEGylated liposomal delivery
23
system. The liposomes (GEM-Lip) were prepared with Lipoid S-100, Cholesterol and
24
DSPE-PEG2000
25
dispersion-extrusion-ammonium sulfate gradient method. The entrapment efficiency
26
(EE, %) and drug loading (DL, %) of the liposomes (117.8 ± 16.85 nm) were 68.83 %
27
and 2.52 %, respectively. The in vitro release study and plasma stability experiments
28
showed the GEM-Lip was more stable than GEM-Sol at pH 7.4 and in the plasma.
29
AUC and Cmax of GEM-Lip were shown to be 6.20 and 16.44-fold higher than that of
(at
a
molar
ratio
of
9:2:0.07)
by
the
film
30
the solutions.30 The pharmacodynamics experiment in Kunming mice bearing a H22
31
cancer cell model showed that the tumor inhibition rate of gemcitabine liposomes was
32
6.25-fold as that of gemcitabine solutions when administrated intravenously. Our
33
findings demonstrated that the preparation of GEM-Lip could effectively enhance the
34
pharmacokinetic properties, the plasma stability and antitumor effect of gemcitabine.
35 36
Key Words：Antitumor effect
Gemcitabine
Liposome Plasma stability
37 38
Scheme 1. Active drug loading mechanism of GEM-Lip and its therapeutic efficacy.
39 40
1.Introduction
41
Gemcitabine (2c, 2c-difluoro-2c-deoxycytidine, GEM) is a deoxycytidine
42
antimetabolite, and has shown excellent efficacy both alone and in combination for
43
the treatment of various malignancies, including ovarian cancer, pancreatic cancer,
44
non-small cell lung cancer as well as other solid tumors [1-3]. The anti-tumor
45
mechanism of gemcitabine is to be absorbed as a prodrug by cells and phosphorylated
46
within the cell to form the active portion of gemcitabine triphosphate, which inhibits
47
DNA synthesis [4, 5]. However, after systemic administration, gemcitabine was
48
rapidly converted into an inactive metabolite by cytidine deaminase and excreted
49
through the urine, which limits its antitumor effect and application in the clinic [6-8].
50
In order to reach a therapeutic level, gemcitabine is currently administered at a high
51
dose of 1,000 mg/m2 for 30 min intravenous (i.v.) infusion, which causes
52
hematotoxicity and other side effects. Therefore, there is a strong need to obtain a
53
preparation of gemcitabine with good plasma stability.
54
In the present studies, some prodrugs of gemcitabine with lipophilic acyl chains
55
were designed to overcome above problems. A lipophilic prodrug can be obtained by
56
linking the 4-amino group of gemcitabine with an acyl chain such as pentanoyl group,
57
heptanoyl group, lauroyl group, stearoyl group or squalenoyl derivative [9]. Studies
58
have shown that prodrugs could enhance the pharmacological activity of the active
59
compound compared to the drug administered in free form [9]. However, further
60
researches, such as safety studies, are still required to prove that there are few side
61
effects of prodrugs. Based on this, encapsulation of gemcitabine into liposomes could
62
be more readily applied into the clinic compared to prodrugs. Encapsulating within
63
liposomes may be a means to protect drugs from metabolic inactivation and reduce
64
the accumulation of drugs in healthy tissues which consequently alleviates the toxicity
65
and adverse effects [10, 11].However, liposomes were usually identificated by the
66
reticuloendothelial system and accumulated less to the target if there is no surface
67
modification which might limit the clinical trial function of gemcitabine. To overcome
68
this type of problem, PEGylated liposomes have been used as carriers for many drugs
69
to achieve a longer cycle time in vivo and have shown great promise for cancer
70
therapy applications [11, 12], notably as Doxil ®. According to the literature, the
71
PEGylated liposomal entrapment of drugs is able to produce a prolonged blood
72
circulation time and facilitate tumor accumulation, therefore effectively promoting the
73
pharmacokinetics (PK) and pharmacodynamics (PD) of the drug and ultimately
74
enhancing the antitumor activity [8]. In addition, studies have shown that GEM
75
liposomes have a significant sustained release in vitro compared with conventional
76
GEM injection [13]. Namely, liposomes could provide protection against rapid
77
metabolic inactivation of drugs, and thereby generate a greater anti-tumor effect in
78
vivo and improve the plasma stability of gemcitabine.
79
Several methods for preparing GEM liposomes (GEM-Lip) have been reported
80
in the literature [14-16], and can be mainly divided into two categories: passive drug
81
loading method (e.g. membrane hydration and reverse phase evaporation) and the
82
active loading method using the ammonium sulfate gradient. When encapsulating a
83
hydrophilic drug such as gemcitabine, the entrapment efficiency (EE%) of the
84
liposome fabricated with passive loading, using the membrane dispersion method and
85
the reverse evaporation method was about 47% [15] and 67% [14], respectively, but
86
the encapsulation conditions were not readily controlled. As well, the particle sizes of
87
the liposomes obtained from these two methods were over 1 µm, which makes it easy
88
to be eliminated by the reticuloendothelial system. However, when instead applying
89
the film dispersion-extrusion-ammonium sulfate gradient method, the liposome
90
particle size could be smaller and more uniform by extrusion and the EE of liposome
91
was slightly higher than above two methods by utilizing the ionic gradient between
92
the inner and outer sides of the phospholipid to form the medicine carrying motive
93
force.
94
According to previous literature [16], the ammonium sulfate gradient method,
95
which is one of the most widely used active loading methods for achieving preferable
96
liposomes, is suitable for loading weak alkaline drugs with pKa ≤ 11 and logP values
97
in the range of -2.5 to 2.0. Therefore, in our study, with a pKa and logP of 3.6 and -1.4,
98
respectively,
99
dispersion-extrusion-ammonium sulfate gradient method to prepare liposome. In
100
addition, by using Lipoid S-100 with little amount of DSPE-PEG2000, this liposome
101
seemed to be more economical than what other literatures reported. So this liposome
102
might be a good choice for gemcitabine to improve its stability in the systemic
103
circulation and antitumor effect.
gemcitabine
might
be
a
suitable
drug
for
the
membrane
104 105
2. Materials and Methods
106
2.1 Materials and Reagents
107
Lipoid E-80 (82% Phosphatidylcholine (PC), 9.2% Phosphatidyl ethanolamine
108
(PE)) and Lipoid S-100 (96% PC, < 0.1% PE) were purchased from Lipoid KG
109
(Ludwigshafen, Germany). PL-100M (78% PC, 18% PE), PC-98T (98.6% PC,< 0.1%
110
PE),and1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy(polyethylene
111
vglycol)-2000]
112
Technology Pharmaceutical Co., Ltd. (Shanghai, China). Cholesterol (CHO) was
(DSPE-MPEG2000) were obtained
from
Advanced
Vehicle
113
provided by J & K (China) and ammonium sulfate was purchased from Shanghai
114
Aladdin Biochemical Technology Co., Ltd (China). Sodium chloride was purchased
115
from Shandong Yuwang Group (China). GEM (HPLC purity >99%) was a kind gift
116
from Shenyang Jin Chang Pharmaceutical Co., Ltd (China). Chloroform, methanol,
117
ethylacetate and isopropanol were provided by Tianjin Concord Technology Co., Ltd.
118
(China). Formic acid was purchased from Thermo Fisher Scientific Inc. (America).
119
All other chemicals and reagents were of analytical or chromatographic grade.
120 121
2.2 Preparation of Gemcitabine-loaded liposomes (GEM-Lip)
122
GEM-Lip was prepared by the thin film dispersion-extrusion-ammonium sulfate
123
gradient method [17, 18]. First, the phospholipids, CHO and DSPE-MPEG2000 were
124
dissolved in a round-bottom flask using chloroform-methanol (3:1 v/v) solution at a
125
molar ratio of 9:2:0.07. The organic solvents were next removed by means of vacuum
126
rotatory evaporator at 40 °C. The resulting lipid film was further dried under a
127
nitrogen stream for 1 h, followed by hydrating and vortexing in 250 mM ammonium
128
sulphate to obtain a suspension of multilamellar vesicles [19]. The suspension was
129
then extruded (LiposoFast LF1, Avestin, Canada) through 100 nm polycarbonate
130
filters (Nucleopore) for 30 times and 50 nm polycarbonate filters for 20 times. To
131
remove the unencapsulated aqueous ammonium sulfate, the products were dialyzed in
132
a 150 mM sodium chloride solution at 35 °C for 6 h. gemcitabine at a concentration of
133
1 mg/mL was then incubated with the blank liposomes at 60 °C for 2 h to obtain the
134
GEM-Lip composite.
135 136
2.3 Characterization of GEM-Lip
137
2.3.1 Particle size analysis
138
The mean particle size and the polydispersity index (PI) of the GEM-Lip was
139
measured by photo correlation spectroscopy (PSC, dynamic light scattering, DLS)
140
with a Nicomp™ 380 particle sizing system (Santa Barbara, USA). Each sample was
141
recorded three times using ten sub-run measurements. To avoid multiple scattering
142
effects, a maximum of 200–300 HZ was adjusted by diluting of the samples with
143
purified water.
144
The morphology of the liposomes was examined by transmission electron
145
microscopy (TEM) (JEM-2100) using negative staining. Briefly, one drop of liposome
146
suspension was put on the top of a 3 mm 200-mesh copper grid. After 2 min
147
incubation, the surplus was removed by filter paper, and the liposome sample on the
148
mesh was stained with 2% phosphotungstic acid solution and incubated for another 2
149
min. The sample was dried at room temperature before use.
150 151
2.3.2 Entrapment Efficiency（ （EE） ）and Drug Loading (DL)
152
The EE of GEM-Lip was determined by centrifugal ultrafiltration. Briefly,
153
200µL GEM-Lip was demulsificated with 1 mL isopropyl alcohol, and diluted with
154
methanol to 10 mL. The total amount of GEM was acquired after ultracentrifugation
155
at 12,000 rpm for 10 minutes and quantified by HPLC in triplicate after filtering
156
through 0.22 µm microporous membranes. Liposomes (1.0 mL) were added to an
157
ultrafilter (Amicon ultra, Millipore Co., USA, MWCO 10 kDa) and centrifuged at
158
3,000 rpm for 15 min. In the ultrafiltrate, unencapsulated drug was obtained and
159
diluted to 10 mL with the mobile phase and quantified as above. HPLC conditions
160
were as follows: a C18 column (250 × 4.6 mm, 5 µm; Thermo Hypersil GOLD aQ,
161
America) was used, the mobile phase was a mixture of 20 mM sodium acetate
162
aqueous-methanol (90:10 [v/v]) at a flow rate of 1 mL/min, the column temperature
163
and wavelength were 25 °C and 268 nm, and the injection volume was 20 µL.
164
EE and DL were calculated using the following equations:
165
EE (%) = (1- WFree / WToal)×100%
166
DL (%) = Wen / WLip×100%
167
where WTotal was the total amount of drug of the preparation and WFree was the
168
amount of drug in ultrafiltrate, respectively. Wen was the amount of drug of entrapped
169
in the liposome, and WLip was the total amount of drug and excipients.
170 171 172
2.4 In vitro drug release study In vitro release of gemcitabine from GEM-Lip was investigated by the dynamic
173
dialysis method. 1 mL of GEM-Lip (n=3) or GEM-Sol (n=3) were placed into a
174
dialysis membrane (molecular weight cut-off 14 kDa, Spectrum Labs, Rancho
175
Dominguez, CA, USA) and incubated in 10 mL of release medium (isotonic pH 7.4
176
PBS, in order to simulate sink conditions in vivo) at 100 rpm under 37 °C with a
177
shaking
178
Manufacturing Co., Ltd, China). At predetermined time intervals (at 0.25, 0.5, 1, 2, 4,
179
6, 8, 12, and 24 h), the medium was withdrawn completely and replaced with 10 mL
180
fresh medium. Samples were then filtered through 0.22 µm filter membrane and
181
analyzed by HPLC to estimate the amount of drug released.
bath
(ZWF-110X30,
Shanghai
Zhicheng
Analytical
Instruments
182 183
2.5 Plasma stability
184
Stability in rat plasma was evaluated by incubating GEM-Sol or GEM-Lip in
185
100 % rat blank plasma at a concentration of 1 mg/mL at 37 ℃ for 24 h. Thereafter
186
50 µL of samples were collected every 2 hours. 20 µL of cytarabine hydrochloride at a
187
concentration of 0.1 mg/mL as an internal standard and 50 µL methanol were added
188
into each sample. Next, 1 mL isopropanol and 2.5 mL ethyl acetate were added into
189
the above mixture and vortexed and centrifuged at 12,000 rpm for 10 min. 3 mL of
190
the supernatant was transferred and evaporated to dryness under a 40 ℃ air stream.
191
The residue was redissloved in 400 µL mobile phase and vortexed for 10 min. The
192
mixture was centrifuged at 12,000 rpm for 10 min and 10 µL of the supernatant was
193
transferred for the content analysis by HPLC. In addition, the plasma stability in terms
194
of particle size of GEM-Lip was also assessed.
195 196
2.6 Pharmacokinetic studies
197
Male SD rats were purchased from Laboratory Animal Center of Shenyang
198
Pharmaceutical University. The rats were randomly divided into two groups (six rats
199
in each group). Each rat was treated with 4 mg/kg of free gemcitabine (GEM-Sol
200
group) or GEM-Lip (GEM-Lip group) through tail vein injection. Blood samples (0.5
201
mL) were removed from the retro-orbital plexus at various times, and then centrifuged
202
at 6,000 rpm for 10 min in a refrigerated centrifuge (Anhui USTC Zonkia Scientific
203
Instruments Co., Ltd, China). The upper plasma samples were stored in a -20 °C
204
freezer until use.
205
For analysis, plasma samples were diluted by adding 1 mL isopropanol and 2.5
206
mL ethyl acetate [20]. After vortex-mixing the samples for 10 min (Lamivudine as
207
internal standard), the mixture was then centrifuged for 10 min at 12,000 rpm. Then 2
208
mL supernatant was transferred and evaporated to dryness under nitrogen stream
209
(40 ℃). The sample residue was redissolved in 400 µL of mobile phase (methanol:
210
0.1% formic acid in water 80:20), and vortexed for 10 min, and then centrifuged at
211
12,000 rpm to obtain the supernatant for analysis by LC-MS / MS. The LC-MS/MS
212
conditions were as follows: The ion source was an electrospray ion source (ESI
213
source positive ion scan). The ion source temperature was 150 °C and the desolvation
214
gas temperature was 400 °C. The desolvation gas flow rate and the cone blowback gas
215
flow rate were 550 L•h-1 and 50 L• h-1. N2 and Ar were used as a solvent removal gas
216
a collision gas, respectively. The scanning method was multiple reactions monitoring
217
(MRM), and the detected m/z values of gemcitabine and lamivudine were
218
264.03/111.92 and 229.92/111.91, respectively. The capillary voltage was 3 kV. When
219
detecting gemcitabine, the cone voltage and collision energy were 20 V and 25 V,
220
respectively. And when the internal standard was detected, the cone voltage and
221
collision energy were 12 V and 10 V.
222 223
2.7 Antitumor activity of GEM-Lip in xenograft animals
224
Animal experiments were carried out in agreement with the principles and
225
procedures outlined by the local Ethical Committee. Mouse liver cancer cells H22 in
226
the exponential growth phase were collected and diluted with sterile saline to adjust
227
the concentration to 1×106 cells/mL. 0.1 mL of the above cell suspension was injected
228
into the peritoneal cavity of Kunming mice. Ascites were extracted under sterile
229
conditions from 7 to 10 days, and diluted to 1×106 cells/mL with sterile saline. The
230
mouse ascites (0.1 mL) were subcutaneously injected in the right flank regions of
231
mice to establish a tumor-bearing mouse model and allowed to grow for 1 week. The
232
Kunming mice were randomly distributed into 3 groups (n=10): saline (Control
233
group), free GEM (GEM-Sol group, 4 mg/kg) or GEM-Lip (GEM-Lip group, 4
234
mg/kg). On the seventh day after the establishment of the mouse tumor model, a
235
considerable dose of preparations or solution was injected into the tail vein every
236
other day for four times, and the control group was injected with 0.2 mL of saline.
237
The tumor volume and body weight of each mouse was measured every day. Tumor
238
volumes were calculated and plotted as average values per group. The sizes of tumor
239
masses were measured with a caliper and tumor volume was calculated according to
240
the formula (Paolino et al. 2010): V = 0.5 × ab2
241 242
where a is the larger perpendicular diameter and b is the smaller perpendicular diameter of the tumor, respectively.
243
The feeding behavior, activity of the mice and motor activity of mice were
244
observed as indicators of general health. The mice were sacrificed by cervical
245
dislocation on the next day after the last administration, and then the tumors were
246
removed and weighed. The tumor inhibition rate (TIR) was calculated as follows: TIR = 1 −
Mean tumor weight of treated group × 100% Mean tumor weight of control group
247
After, the tumor masses were eradicated and rinsed in saline for analysis. They
248
were fixed in 4 % (w/v) buffered formaldehyde (pH = 7.4) at room temperature,
249
dehydrated in alcohol and then embedded in paraffin. Sections with a thickness of 7–
250
10 µm were sliced using a microtome and stained following the eosin B / hematoxylin
251
method [21] (HE staining). The sections were then subjected to histopathological
252
examination under a microscope.
253 254
2.8 Statistical analysis
255
All experiments were performed at least three times and expressed as means ±
256
SD and results were dealed with Microsoft Excel 2010 and Graphpad prism 6. Data
257
were analyzed for statistical significance using Student’s test. p < 0.05 was considered
258
statistically significant, and p < 0.01 was considered highly significant.
259 260
3. Results
261
3.1 Characterization of gemcitabine loaded liposomes (GEM-Lip)
262
The EE of the liposomes was approximately 68.83 % with a final DL of
263
approximately 2.52 %. The particle size was around 117.8 ± 16.85 nm with a PDI of
264
0.020. The morphology of the particles was characterized by TEM in Fig.1.
265 266
Fig.1.TEM images of GEM-Lip (A and B), the particle size distribution of GEM-Lip
267
(C) and the structure of GEM-Lip (D).
268 269
3.2 In vitro release studies
270
A drug release study was performed to evaluate the drug release pattern and form
271
of drug release. The drug release was represented graphically by plotting percent drug
272
release against time, and the results are depicted in Fig.2. The cumulative release
273
percentage of the liposomes was 12.48 % at 1 h, whereas a quick diffusion of
274
GEM-Sol was observed at the same time point. By fitting the release curve to the
275
release model, it was found that the release of gemcitabine liposomes was in line with
276
the Ringer-Peppas model.
277 278
Fig.2. GEM-Lip and GEM-Sol were released in PBS at pH 7.4 (mean ± SD, n=3)
279 280
3.3 Plasma stability
281
As shown in Fig.3B, the percentage of drug remaining for GEM-Sol was about
282
20 % after incubating with rat plasma for 2 h, while that for GEM-Lip was above
283
90 %. The percentage of drug remaining was stable when incubating about 8 h, and
284
that for this two preparations were below 5 % (GEM-Sol) and above 50 % (GEM-Lip)
285
(p < 0.01) , respectively. And the particle size of GEM-Lip remained stable at 80 - 110
286
nm after incubating 8 h (p ＞ 0.05), which was consistent with the result of the
287
percentage of drug remaining.
288
Fig.3. The stability of particle size (A) and the percentage of drug remaining of
289
GEM-Lip (B) after incubating in the rat plasma for 24 h. Asterisks indicate significant
differences (t test; *p<0.05; **p<0.01)
290 291 292
3.4 Pharmacokinetics studies
293
In this experiment, the LC-MS / MS method was selected to determine the
294
plasma concentration of GEM-Lip and GEM-Sol. The mean gemcitabine plasma
295
concentration–time profiles after intravenous administration at a dose of 4 mg•kg-1 (n
296
= 6) of GEM-Sol and GEM-Lip are shown in Fig.4, and the important PK parameters
297
are listed in Table 1. As shown in Table 1, the AUC
298
group were 6.54 and 6.20-fold higher than that of the GEM-Sol group, respectively.
299
The t1/2z of GEM-Lip was around 2.89 h. The Vz and CLz of GEM-Lip were largely
300
decreased, and were 0.26 and 0.16-fold lower than that of the solutions. In addition,
301
the Cmax of GEM-Lip was nearly16.44-fold as that of GEM-Sol.
(0-t)
and AUC (0-∞) of GEM-Lip
302 303
Fig.4. Concentrations of GEM in male rat plasma after a single i.v. treatment (mean ±
304
SD, n = 6)
305 306
Table1. The Pharmacokinetics parameters of GEM-Sol and GEM-Lip Parameters
GEM-Sol
GEM-Lip
AUC(0-t)( ug/L*h)
2267.8 ± 273.47
14824.66 ± 467.60
AUC(0-∞)( ug/L*h)
2394.6± 353.67
14849.59 ±502.49
Cmax(ug/L)
608.9 ± 20.49
10010.3 ±1067.46
CLz(L/h/kg)
1.70 ± 0.22
0.27 ± 0.009
Vz(L/kg)
4.27 ± 0.73
1.11 ± 0.36
t1/2z(h)
1.77 ± 0.38
2.89 ± 1.04
307 308
3.5 Antitumor activity studies in mice
309
As shown in A and B of Fig.5, after 4 treatment doses, the volume of the tumors
310
in the mice treated with saline was 3329 mm3 and that with GEM-Sol was 2621 mm3,
311
while those of mice treated with GEM-Lip were smaller (∼1699 mm3). No evident
312
change in body weight was observed in the GEM-Lip group throughout the week of
313
study, however there was a slight drop in body weight for the control group compared
314
with other treatments. The results are expressed as an average, and T-tests were used
315
to determine statistical significance with p < 0.05 considered statistically significant
316
[22]. There were also differences in the survival status of mice in each administration
317
group. At the end of the administration period, four mice died in the control group due
318
to the deterioration of the condition and two died in the GEM-Sol group and
319
GEM-Lip group, and tumors of a mouse in this group were not obviously observed.
320
When comparing GEM-Lip and GEM-Sol, containing the same dose of gemcitabine,
321
the drug-loaded liposomes had an improved antitumor effect, and there was also a
322
significant difference compared with the normal saline group. The weight of the
323
collected tumor masses shown in Fig.5C confirmed these findings. Masses of ∼0.48 g
324
were observed in the case of GEM-Lip, while the weight of tumors in the control
325
group and the GEM-Sol group were ~1.23 g and ~1.11 g, respectively. Fig.5D
326
expresses the above differences. The tumor inhibition rate (TIR) of GEM-Sol was
327
9.76% and that of GEM-Lip was 60.98% (p < 0.05).
328
As shown in Fig.6, when the tumor slices were observed under microscope, a
329
large number of tumor cells were observed in the control group. In the GEM-Sol
330
group, tumor cells were deeply stained and nuclear pyknosis appeared, and necrotic
331
tumor cells were observed in the middle. In the GEM-Lip group, there were also
332
deeply stained, large, and multinucleated tumor cells, but the tumor cells in the
333
GEM-Lip group appeared to be relatively sparse comparing with the control group.
334
Fig.5. Tumor volume changes (A) , body weight changes (B) of various preparations
335
after i.v. injection in the tail, solid tumors stripped from H22-tumor-bearing mice(C)
336
and the tumor weight of different groups (D)
337 338
Fig.6. Histological analysis of mouse tumor excised of Control group (A), GEM-Sol
339
group (B), and GEM-Lip group (C) at the end of experiment
340 341
4. Discussion
342
As a major antitumor drug, gemcitabine has been widely applied in the treatment
343
of various tumors. However, it is easy to be metabolized by cytidine deaminase limits
344
its
antitumor
effect.
The
liposome
produced
by
the
film
345
dispersion-extrusion-ammonium sulfate gradient method might protect gemcitabine
346
from being metabolism. When GEM-Lip was prepared by film dispersion method, a
347
low EE (<30%) was obtained. And instability of particle size made it difficult to apply
348
reverse
349
dispersion-extrusion-ammonium sulfate gradient method might be superior to above
350
methods to a certain extent in our experiments. On one hand, particle size uniformity
351
(about 100 nm) could be guaranteed by extrusion. On the other hand, the role of
352
ammonium sulfate in the internal aqueous phase of the liposome was to provide an
353
acidic environment to elicit the protonation of gemcitabine, and thereby, distinctly
354
reduced the drug leakage from the liposome to a certain degree. In addition, due to the
355
difference of pH between the internal and external of liposome, there was a driving
356
force for unionized moiety to enter the internal of the liposome and be ionized.
357
According to the equilibration formula between the ionized and unionized
358
gemcitabine inside the liposome, the ratio of the amount of ionized and unionized part
359
was 2:1 when the equilibration was obtained [16]. So the max EE theoretically was
360
about 66.67%. Obviously, by applying the film dispersion-extrusion-ammonium
361
sulfate gradient method, the max EE was obtained in this experiment.
evaporation
method
to
the
final
preparation.
While
film
362
The in vitro release study was conducted at pH 7.4 to mimic the physiological
363
pH. The release profiles showed that gemcitabine in the liposomes could release more
364
slowly than in aqueous solution. A quick diffusion was observed in the GEM-Sol
365
group. However, the release profile of the liposomes was S-type, which suggested that
366
there was a fast release of GEM-Lip. And this might be due to the osmotic pressure
367
difference between the internal (550 mOsm) and external liposome (200 mOsm),
368
which caused it swelling with the influx of water [16]. In addition, the resistance of
369
the major release course mainly diffusion from the liposome inner water phase, so a
370
slow release phenomenon was observed next. Above results indicated that the
371
liposomes could be more stable than solutions at the physiological pH.
372
The plasma stability studies also confirmed above conclusion. The stability of
373
GEM-Lip was greater than GEM-Sol in rat plasma, which may be due to the
374
encapsulation of gemcitabine in the liposome and the protonation of gemcitabine
375
inside the liposome could reduce its exposure to plasma and prevent it from being
376
metabolized by cytidine deaminase, thus improving its stability in the rat plasma.
377
However, the particle size of GEM-Lip was decreasing with time and was stable from
378
8 h (p ＞ 0.05) which showed in the Fig 3A, and this indicated that the GEM-Lip
379
could maintain its structure when it existed in the blood.
380
The pharmacokinetics studies also verified the better stability of the liposomes.
381
As reported in literature, GEM-Sol was usually administered with larger doses in the
382
clinic to achieve therapeutic effects. This can cause many adverse effects such as
383
myelosuppression and impaired liver function. In this experiment, as stated above, the
384
plasma stability of GEM-Lip was confirmed to be better than GEM-Sol, thus the
385
preparation of GEM-Lip could be expected to reduce the required drug dose and
386
improve its antitumor effect. PEGylated liposomes could help gemcitabine circulating
387
for longer time in the systemic circulation and protected it from being metabolized. In
388
our experiment, gemcitabine was basically all encapsulated in the liposome after tail
389
vein injection, but GEM-Sol was unfortunate to meet the cytidine deaminase
390
immediately and was metabolized. So the Cmax of GEM-Lip was extremely higher
391
than GEM-Sol. However, as the time going on, GEM-Lip was constantly striking the
392
materials in the bloodstream while gradually cracked and gemcitabine in the inner
393
phase of liposome was exposed to the cytidine deaminase and was metabolized
394
rapidly, which caused the short t1/2. As well, other PK parameters of the liposome
395
which were compared with GEM-Sol confirmed that the encapsulation of GEM in
396
liposomes could limit the distribution range of drug in the systemic circulation[23].
397
For instance, the Vz and CLz of the liposomes were lower than the solutions, which
398
could reduce the possibility of active agent metabolism. Specifically, the lower Vz can
399
be an indication of reducing toxic side effects, and the lower CLz can represent the
400
long system cycle time and high stability of GEM-Lip. The t1/2z and Cmax of the
401
liposomes were longer and greatly larger respectively than the solutions, and this
402
indicated that the active agent could be stable in the blood for a longer time. All above
403
parameters confirmed that the GEM-Lip could protect gemcitabine from being
404
metabolized by cytidine deaminase.
405
The results of pharmacodynamics experiment showed the excellent antitumor
406
effect of the liposomes. The tumor volume and tumor weight of tumor-bearing mice
407
of the liposomes group were smaller than GEM-Sol group. The survival status of mice
408
in the liposomes group was also better than other groups. As Laquente studied, the
409
conventional dose of gemcitabine for mice was 100 mg/kg, and was administered on
410
days 0, 3, 6 and 9. And the TIR of the conventional schedule of gemcitabine was
411
about 97.59 % to human pancreatic carcinoma [24]. However, with so high treatment
412
dose, some side effects such as myelosuppression might be extremely harmful to
413
patients. Compared to this, though the TIR of GEM-Sol was extremely lower than
414
conventional dose, this lower treatment dose might cause fewer side effects and
415
relieve the suffering of patients. In addition, the TIR of GEM-Lip was 60.98 % even
416
with so low treatment dose.
417
These phenomenon might be because it always needs a high treatment dose for
418
gemcitabine to achieve the treatment effects. While the treatment dose of GEM-Sol
419
and GEM-Lip group in this study was 4 mg/kg, which was much lower than
420
conventional dose of gemcitabine. So the TIR of GEM-Sol was extremely lower than
421
expected. However, due to the protection of PEGylated phospholipids and the
422
encapsulation of gemcitabine, it was protected from cytidine deaminase metabolism
423
to a certain extent, thereby the body cycle time of gemcitabine was prolonged and the
424
AUC was increased. In addition, the presence of hydrophilic chain of
425
DSPE-MPEG2000 on the surfaces of liposomes was important for preventing their
426
uptake by the reticuloendothelial system and consequently for increasing the t1/2z of
427
liposomes and their prolonged presence in the bloodstream. In summary, the
428
antitumor activity of GEM-Lip was enhanced compared with GEM-Sol.
429 430
5. Conclusion
431
In this study, a GEM-Lip with a uniform particle size and high entrapment
432
efficiency was prepared by film dispersion-extrusion-ammonium sulfate gradient
433
method. The in vitro studies showed that the GEM-Lip could be more stable than
434
GEM-Sol at the physiological pH and in the rat plasma. The in vivo studies confirmed
435
that the GEM-Lip could protect gemcitabine from being metabolized by cytidine
436
deaminase. Furthermore, the pharmacodynamics results demonstrated that the
437
GEM-Lip could enhance antitumor activity effectively. In conclusion, the
438
encapsulation of gemcitabine in the liposome could improve its stability in the rat
439
plasma and enhance its antitumor effect to a certain degree.
440 441
Acknowledgments
442
We sincerely thank Amanda Pearce for the linguistic assistance during the
443
revision of this manuscript. There are no conflicts of interest within the authors. In
444
addition, the authors are very grateful to the 2016 Annual Youth Teachers’ Career
445
Development Support Program of Shenyang Pharmaceutical University [grant number
446
ZQN2016008].
447 448
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Declarations of interest: There was no conflict of interest among all authors.