Simple weak-acid derivatives of paclitaxel for remote loading into liposomes and improved therapeutic effects

Article abstract of DOI:10.1039/d0ra03190a

Liposomes are among the most successful nanocarriers; several products have been marketed, all of which were prepared by active loading methods. However, poorly water-soluble drugs without ionizable groups are usually incorporated into the lipid bi-layer

Full text of DOI:10.1039/d0ra03190a

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Simple weak-acid derivatives of paclitaxel for
remote loading into liposomes and improved
Cite this: RSC Adv., 2020, 10, 27676
therapeutic effects†
Jiang Yu,‡a Shuang Zhou,‡a Jinbo Li,a Yingli Wang,a Yujiao Su,a Dongxu Chi,b
a
c
d
Jiamei Wang,a Xue Wang,b Zhonggui He,
Guimei Lin, Dan Liu
*
a
*
and Yongjun Wang
Liposomes are among the most successful nanocarriers; several products have been marketed, all of which
were prepared by active loading methods. However, poorly water-soluble drugs without ionizable groups
are usually incorporated into the lipid bi-layer of liposomes by passive loading methods, with serious drug
leakage during blood circulation. Furthermore, there have been few improvements in their anti-cancer
activity and safety. Herein, we designed and synthesized three weak-acid modified paclitaxel (PTX)
derivatives with a one-step reaction for the remote loading of liposomal formulations. By comparison,
PTX-succinic acid liposomes (PTX-SA LPs) exhibited the highest encapsulation efficiency (97.2 ꢀ 1.8%)
and drug loading (8.84 ꢀ 0.16%); meanwhile, there was almost no change in their particle size or zeta
potential within one month. Furthermore, compared with Taxol®, the PTX-SA LPs showed a 4.35-fold
prolonged half-time, enhanced tumor accumulation, and an increased maximum tolerated dose (MTD)
of more than 30 mg kgꢁ1. As a result, the PTX-SA LPs displayed significantly improved in vivo anti-cancer
efficacy in comparison with Taxol®. Therefore, weak-acid modification is proved to be a simple and
effective method to achieve remote loading and high encapsulation efficiency of poorly soluble drugs,
showing great potential for clinical application.
Received 9th April 2020
Accepted 27th May 2020
DOI: 10.1039/d0ra03190a
rsc.li/rsc-advances
suitable for drugs that are water-soluble and contain weakly
acidic or alkaline groups. In addition to the traditional pH
1. Introduction
gradient, the ammonium ion gradient is oen used as the
transmembrane driving force of weakly basic drugs,4,5 while
weakly acidic drugs usually adopt the acetate ion gradient.2,6
Liposomes prepared by ammonium/acetate ion gradients can
stabilize drugs in the intraliposomal aqueous phase by forming
impermeable complexation or precipitation with counter ions
in order to avoid the leakage of drugs.6,7 Several liposomal
formulations prepared by active loading methods have been
approved by the FDA for clinical cancer therapy, such as
Doxil®,8,9 Onivyde®,10,11 Marqibo®,11 and Vyxeos®.12 However,
poorly water-soluble drugs are normally incorporated into the
lipid bilayers of liposomes with limited drug loading capacity
and poor drug retention, resulting in few improvements in anti-
cancer activity and safety.13,14
Liposomes are an efficient nano-drug delivery system (nano-
DDS) for cancer therapy; they have the advantages of good
biocompatibility and biodegradability, non-toxicity, and sus-
tained drug release.1–3 Drugs can be loaded into liposomes by
active or passive loading methods. The active loading method
refers to loading drugs by the driven force of an ion/compound
gradient between the intra- and extra-liposomal aqueous pha-
ses. In the passive loading method, drugs are rst dissolved in
water or an organic phase; then, drug-containing liposomes are
prepared by suitable methods. Generally, active drug loading is
aWuya College of Innovation, Shenyang Pharmaceutical University, Shenyang,
Liaoning, 110016, P. R. China. E-mail: wangyongjun@syphu.edu.cn; Fax: +86-24-
23986325; Tel: +86-24-23986325
Appropriate amphiphilicity (partition coefficient, log P) and
acid-basicity (dened by their pKa) of drugs are necessary for
successful remote loading; this limits the use of many rst-line
chemotherapeutic drugs for active loading liposomal formula-
tions.5,15 In the past decades, chemical modication strategies
have been developed by introducing an ionizable functional
group and improving the membrane permeability of drugs for
remote loading. Irinotecan is one of the most successful
examples; its marketed liposomal formulation, Onivyde®, was
bDepartment of Pharmaceutics, Shenyang Pharmaceutical University, Shenyang,
Liaoning, 110016, P. R. China
cSchool of Pharmaceutical Science, Shandong University, 44 Wenhuaxi Road, Jinan,
250012, China
dKey Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education,
Shenyang Pharmaceutical University, Shenyang 110016, Liaoning, P. R. China. E-mail:
sammyld@163.com; Fax: +86-24-4352-0218; Tel: +86-24-4352-0218
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra03190a
‡ Both authors contributed equally.
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modied based on SN-38 with
a
weakly basic 4-piper- efficiency (97.2 ꢀ 1.8%) and drug loading (8.84 ꢀ 0.16%). Thus,
idinopiperidine group via ester bonds.16 In addition, a hydro- the PTX-SA LPs were chosen for the following experiments. The
phobic drug, docetaxel, was encapsulated into the PTX-SA LPs displayed excellent stability and sustained drug
intraliposomal aqueous phase by chemically modifying release in vitro. Moreover, the PTX-SA LPs demonstrated 4.35-
a piperazine or tertiary amine group at the C-20 position using fold-prolonged drug half-time and an area under the curve
an ammonium sulfate (AS) or triethylamine sucrose-octa-sulfate (AUC) enhanced by more than 200 times compared to that of
(TEA-SOS) gradient.17–19 Gemcitabine (GEM), a highly water- free PTX. Furthermore, the PTX-SA LPs displayed improved in
soluble drug with poor membrane permeability, is normally vivo anti-cancer efficacy in comparison with Taxol®. Therefore,
prepared as a liposomal formulation via a passive loading the PTX-SA LPs are proved to be a more effective formulation,
strategy with low encapsulation efficiency and a low drug-to- with negligible toxicity to normal tissues.
lipid ratio.20,21 Considerable improvement was accomplished
in the remote loading of GEM by introducing a weak base
moiety at the N4 position, and the prepared liposomes
possessed high drug-to-lipid ratios and good stability.15 2.1 Synthesis of PTX-SA, PTX-GA and PTX-DA
2. Results and discussion
Although great progress has been made, there are still compli-
cated steps in the synthesis of some derivatives, and the
potential toxicity of the modied groups is unclear.
Three weak-acid modied PTX derivatives, PTX-SA, PTX-GA and
PTX-DA, were successfully synthesized by conjugating PTX with
succinic acid (SA), glutaric acid (GA) and trans-2-butene-1,4-
dicarboxybic acid (DA) via ester bonds, respectively (Chart 1).
Paclitaxel (PTX), a rst-line chemotherapeutic drug, has
been widely utilized to treat multiple types of tumors in the
It has been reported that SA can play important roles in the
clinic.22–24 The rst commercial formulation of PTX, Taxol®, was
elds of immunology and cancer, which deserves more detailed
formulated with poly-oxy-ethylated castor oil (CrEL) and dehy-
studies.32,33 Additionally, SA is a biocompatible organic acid
drated ethanol at a ratio of 1 : 1 (v/v).25,26 However, Taxol® is
because it is an intermediate metabolite in the tricarboxylic acid
associated with a series of serious adverse pharmacological and
(TCA) cycle. This can ensure that the degradation product is
non-toxic to the body. Thus, SA was chosen for the synthesis of
the weak-acid derivative of paclitaxel. Meanwhile, for compar-
toxicological effects, which limits its clinical application.27,28 In
addition, a liposomal PTX formulation, Lipusu®, has been
marketed in China, and some other liposomal formulations are
ison, GA and DA were also chosen in order to investigate the
in clinical trials.29–31 However, the loading capacities for these
inuences of the carbon chain length and double bonds on the
encapsulation efficiency and stability of the liposomes. The
formulations are all less than 5%.31 Furthermore, all of them are
prepared by passive loading methods, with poor drug retention.
chemical structures of PTX-SA, PTX-GA and PTX-DA were veri-
ed by H nuclear magnetic resonance (1H NMR) spectra and
From this perspective, it is essential to develop a novel formu-
lation of PTX with high drug loading as well as high delivery
efficiency.
Based on the problems mentioned above, we designed and
synthesized three simple weak-acid-modied PTX derivatives,
PTX-succinic acid (PTX-SA), PTX-glutaric acid (PTX-GA), and
PTX-trans-2-butene-1,4-dicarboxylic acid (PTX-DA) with one-step
reactions, and we attempted to encapsulate them into lipo-
somes by remote loading (Scheme 1). Aer screening, the PTX-
SA liposomes (PTX-SA LPs) exhibited the highest encapsulation
1
mass spectra (MS). The 1H NMR spectra and MS results are
shown in Fig. S1–S3.† The results showed that the yields of PTX-
SA and PTX-GA were higher than that of PTX-DA. This can be
attributed to the higher reactivity of anhydride than of carbox-
ylic acid. Thus, in order to increase the yield, the carboxyl group
of DA must be activated in an ice bath rst. Moreover, more by-
products were generated in the PTX-DA reaction. It was difficult
to separate the targeted product by pre-HPLC methods. Thus,
column chromatography was used.
Scheme 1 The diagram for the preparation of remote loading liposomes and their application in cancer therapy.
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LPs, correspondingly. However, the PTX-GA LPs were aggre-
gated, with a 41.8 ꢀ 2.6% encapsulation efficacy of the drug.
This can be attributed to the inappropriate acid strength and
membrane permeability of PTX-GA. The EE (%) of the PTX-DA
LPs was 86.9 ꢀ 3.4%. However, the PTX-DA LPs turned cloudy
ꢂ
aer storage at 4 C for a week. Thus, the chemical stability of
PTX-DA was investigated, and the results showed that the
compound PTX-DA was degraded by more than 30% within one
month. We speculated that this is due to the unsaturated
double bond, which accelerated the degradation of PTX-DA. The
PTX-SA LPs exhibited the highest EE (%) of 97.2 ꢀ 1.8% with
good stability. Therefore, the PTX-SA LPs were selected for
further evaluations. The specic characterization of the PTX-SA
LPs is shown in Fig. 1B–D; the particle size, PDI and zeta
potential of the PTX-SA LPs are 127.3 ꢀ 2.9 nm, 0.065 ꢀ 0.035
and ꢁ0.039 ꢀ 0.226 mV, respectively. The TEM micrograph
demonstrates that the PTX-SA LPs are nearly spherical and that
the particle size is close to the results measured by DLS (Fig. 1E).
On the other hand, we also attempted to use non-gradient
liposomes to load PTX-SA. However, the drug precipitated aer
drug loading (Fig. S4†) and the amount of drug encapsulated in
the liposomes was lower than the detection limit, indicating that
the process of drug loading was dependent on the calcium acetate
gradient. All these results demonstrate that the high EE of PTX-SA
LPs can be attributed to the calcium acetate gradient, which
remotely entrapped the drug into liposomes rather than only being
incorporated into the lipid bilayer by hydrophobic interactions.
Chart 1 Chemical structures of the weak-acid modified PTX deriva-
tives: PTX-SA, PTX-GA, and PTX-DA.
2.2 Preparation and characterization of liposomes
2.2.1 The principle of remote drug loading. The acetate ion
gradient is oen used as the transmembrane driving force for
weakly acidic drugs. Due to the different permeability coeffi-
cients of acetic acid and calcium ions, the calcium ions are
entrapped inside the liposomes and the acetic acid acts as
a proton shuttle, thus creating a pH gradient (high inside) to
encapsulate the weakly acidic drug into liposomes.1 Taking the
remote loading of PTX-SA as an example, PTX-SA was entrapped
2.3 Physical stability
in the aqueous core of liposomes using a calcium acetate The long-term stability and plasma stability of the PTX-SA LPs were
gradient with the assistance of the organic solvent DMSO. It was evaluated. As shown in Fig. 2A and B, aer storage at 4 ꢂC for one
reported that a limited amount of DMSO can not only improve month, the particle size, PDI and zeta potential of the PTX-SA LPs
the solubility of poorly soluble drugs but can also enhance the showed no obvious changes. Additionally, the PTX-SA LPs were
permeability of liposomal membranes, thus facilitating the incubated in PBS (pH 7.4) containing 10% FBS under 100 rpm at
remote loading process (Fig. 1A).34,35
37 ꢂC for 48 h, and the particle size and PDI of the PTX-SA LPs were
2.2.2 Characterization of liposomes. Typically, the thin- nearly unchanged as well (Fig. 2C). All these data proved that the
lm hydration method is applied to prepare blank Ca(Ac)2 PTX-SA LPs exhibit excellent stability, which can be benecial for
liposomes. The drugs were dissolved in DMSO and were then long circulation and tumor accumulation in vivo aer injection.
added dropwise to the blank gradient liposomes under
ꢂ
continuous stirring at 65 C. When the drug-containing DMSO 2.4 In vitro drug release
solution was added to the blank-gradient liposomes, the solu-
tion initially became cloudy. As the drug loading proceeded, the
solution changed from cloudy to clear. This phenomenon was
similar to that of carlzomib.36 We speculated that there is
a solubility equilibrium of the drug in the extra-liposomal
aqueous phase. The dissolved drug could enter the intra-
The in vitro drug release of the PTX-SA LPs was studied under the
conditions of PBS (pH 7.4) containing 0.5% Tween 80 (v/v). As
shown in Fig. 3, the PTX-SA LPs only released about 25% PTX-SA
and 3% PTX in total within 24 h, indicating an obviously sustained
release rate. Meanwhile, compared with other conventional active
loading liposomes, the PTX-SA LPs exhibited a faster drug release
rate. This can be attributed to the different state of the drug inside
liposomal aqueous phase driven by the transmembrane
driving force. In this process, Ca2+ can also play an important
the liposomes, which merits further investigation.
role. Once the drug enters the liposomes, it can bind with Ca2+
to prevent leakage, thus constantly facilitating the rapid disso-
lution of the drug outside the liposomes and the process of drug
loading until all the drug is dissolved and entrapped.2
The characterization results of the drug-loaded liposomes
are shown in Table 1. The drug-to-lipid ratio for drug loading
was 1/10 (w/w). Aer drug loading, there were no obvious
changes in particle size or PDI for the PTX-SA LPs and PTX-DA
2.5 Cytotoxicity assay
The in vitro cytotoxicities of PTX solution, PTX-SA solution and
PTX-SA LPs against breast cancer cells (4T1) and prostatic
cancer cells (RM-1) were evaluated via MTT viability assay. The
half maximal inhibitory concentrations (IC50) were calculated
and are summarized in Table 2. The IC50 values for PTX solu-
tion, PTX-SA solution and PTX-SA LPs against RM-1 cells at 48 h
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Fig. 1 Characterization of remote loading liposomes. (A) PTX-SA as an example for the mechanism of remote loading with a calcium acetate
gradient. (B) The particle size and (C) zeta potential of the PTX-SA LPs. (D) Photograph of the PTX-SA LPs. (E) TEM image of the PTX-SA LPs.
were 61.33, 202.9 and 272.8 ng mLꢁ1, respectively. However, 4T1
2.6 Pharmacokinetic study
cells appear to be more sensitive to the preparations than RM-1
The pharmacokinetic proles of Taxol® and PTX-SA LPs in SD
cells, with a correspondingly lower IC50
.
rats were evaluated following intravenous administration. The
pharmacokinetic parameters were calculated and are summa-
rized in Table 3, and the pharmacokinetic curves of PTX-SA and
As shown in Fig. 4A–D, the cell viability exhibited time and
concentration dependence. The cytotoxicity of PTX-SA solution was
lower than that of PTX solution, which can be attributed to the fact
that PTX-SA requires more time to release PTX. In addition, the
PTX-SA LPs demonstrated reduced cytotoxicity in contrast to PTX-
SA solution at 48 h. Meanwhile, the IC50 values of PTX-SA solution
and the PTX-SA LPs were very close to each other at 72 h. We
speculated that this is due to the sustained release of the PTX-SA
LPs. On the other hand, due to the incorporation of DSPE-PEG,
the cellular uptake of the PTX-SA LPs may be hindered in some
degree compared with that of the PTX-SA solution. In short, the
PTX-SA LPs could efficiently suppress the proliferation of tumor
cells, showing great anti-cancer potential.
Table 1 Characterization of the PTX-SA LPs (mean ꢀ SD, n ¼ 3)a
Formulation
Size (nm)
PDI
EE (%)
Blank LPs
PTX-SA LPs
PTX-DA LPs
125.9 ꢀ 2.8
127.3 ꢀ 2.9
130.4 ꢀ 2.4
0.084 ꢀ 0.039
0.065 ꢀ 0.035
0.035 ꢀ 0.012
—
97.2 ꢀ 1.8
86.9 ꢀ 3.4
a
EE: encapsulation efficiency.
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Fig. 2 Physical stability of the PTX-SA LPs. (A) The particle size, PDI and (B) zeta potential variation of the PTX-SA LPs at 4 ꢂC for 30 days, (C) the
particle size and PDI of PTX-SA LPs incubated in PBS containing 10% FBS (v/v) under 100 rpm shaking at 37 ꢂC. Data are presented as mean ꢀ SD
(n ¼ 3).
PTX are shown in Fig. 5. Due to the rapid elimination of Taxol®, derived from PTX-SA LPs increased over time up to 48 h in
the concentration of PTX was below the limit of detection at tumors and was higher than Taxol® at 48 h. This can be
24 h post injection (<10 ng mLꢁ1), indicating a short half-time. attributed to the good stability and prolonged blood circulation
It was reported that the commercial liposomal formulation time of the PTX-SA LPs, which in turn facilitates the accumu-
Lipusu® exhibited a 2-fold higher half-time and an over 2.5-fold lation of liposomes at the tumor site by the EPR effect. Overall,
greater area under the curve (AUC) than Taxol®.31 In contrast, the PTX-SA LPs exhibited higher tumor accumulation compared
the half-time and AUC of the PTX-SA LPs were signicantly with Taxol®. Thus, we speculate that the PTX-SA LPs can display
improved by 4.35-fold and 212.8-fold in comparison with those more potent anti-cancer activity than Taxol® in vivo.
of Taxol®, respectively. This can be ascribed to the excellent
stability of the PTX-SA LPs, which reduces leakage of the drug
during blood circulation. All the data demonstrate that the PTX-
SA LPs can noticeably prolong blood circulation time, which in
turn can increase tumor accumulation.
2.8 In vivo antitumor efficacy study
Encouraged by the outstanding results of the in vitro and in vivo
studies, the in vivo anti-cancer efficacies of Taxol® and PTX-SA
LPs were further evaluated in BALB/c mice bearing 4T1 xeno-
gra models (Fig. 7A). Mice with tumor volumes around 100
mm3 were randomly divided into four groups, and PBS, Taxol®
(8 mg kgꢁ1), PTX-SA LPs (equivalent of 8 mg kgꢁ1 or 30 mg kgꢁ1
PTX) were administrated intravenously every three days for
a total of four injections. As shown in Fig. 7B, at the end of the
experiment, the tumor volume of the PBS group was around
1100–1300 mm3. In comparison, the other three groups dis-
played different levels of tumor inhibition aer treatment.
There have been reported that no signicant difference was
observed between the anti-cancer efficacy of Taxol® and that of
commercial liposomal paclitaxel (Lipusu®).31 By contrast, PTX-
SA LPs (8 mg kgꢁ1) exhibited more signicant tumor inhibition
efficacy than Taxol® (P < 0.001), which can be ascribed to the
good stability of the PTX-SA LPs in blood circulation and higher
accumulation at the tumor site. Moreover, the high-dose PTX-SA
LPs (30 mg kgꢁ1) were more effective than the low dose
formulation (8 mg kgꢁ1) (P < 0.05), with only a slight increase of
tumor volume.
2.7 In vivo biodistribution study
With the promising extension of blood circulation, 4T1 tumor-
bearing BALB/c mice were used to evaluate the biodistribution
of Taxol® and the PTX-SA LPs. The concentrations of PTX-SA
and PTX at the major organs and tumors were detected by
UPLC-MS-MS. As shown in Fig. 6A–E, the drugs that accumu-
lated in normal tissues were rapidly eliminated with time. By
contrast, the distribution of PTX-SA LPs at the tumor site was
nearly identical from 6 h to 24 h, and there was a slight decrease
at 48 h (Fig. 6D). Furthermore, it is noteworthy that the PTX
Table 2 IC50 values (ng mLꢁ1) of PTX, PTX-SA and PTX-SA LPs against
4T1 and RM-1 cells (mean ꢀ SD, n ¼ 3)
4T1
RM-1
48 h
Formulation
48 h
72 h
72 h
PTX
PTX-SA
PTX-SA LPs
48.9 ꢀ 1.7
16.3 ꢀ 1.2
61.5 ꢀ 1.8
98.3 ꢀ 2.0
61.3 ꢀ 1.8
202.9 ꢀ 2.3
272.8 ꢀ 2.4
20.79 ꢀ 1.3
40.2 ꢀ 1.6
43.3 ꢀ 1.6
Fig. 3 The in vitro release of PTX-SA and PTX from the PTX-SA LPs in
the medium of PBS (pH 7.4) containing 0.5% Tween 80 (v/v) at 37 ꢂC.
Data are presented as mean ꢀ SD (n ¼ 3).
92.7 ꢀ 2.0
205.8 ꢀ 2.3
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Fig. 4 In vitro cytotoxicity of PTX-SA LPs. Viability of cells incubated with a series of concentrations of PTX, PTX-SA and PTX-SA LPs against 4T1
cells for 48 h (A) and 72 h (B) and against RM-1 cells for 48 h (C) and 72 h (D). Data are presented as mean ꢀ SD (n ¼ 3).
Additionally, the safety of the PTX-SA LPs was investigated by transaminase (ALT), blood urea nitrogen (BUN), and creatinine
monitoring body weight, measuring four biochemical criteria of in the blood of the mice were detected to evaluate their hepatic
blood, and H&E staining tissue slices. During the experiment, and renal function. As shown in Fig. 7E, there were no
there was no signicant variation in body weight in the high-
dose group, indicating that PTX-SA LPs could remarkably
improve the maximum tolerated dose (MTD) (Fig. 7C). However,
when Taxol® was administrated with a dosage of 30 mg kgꢁ1
,
the body weight of the mice was reduced by around 27%,
demonstrating extremely serious side effects (Fig. S5†). The
levels of aspartate transaminase (AST), serum alanine
Table 3 The major pharmacokinetic parameters of Taxol® and the
PTX-SA LPs (mean ꢀ SD, n ¼ 3)
Taxol®
PTX
PTX-SA LPs
Parameter
PTX
PTX-SA
T1/2 (h)
2.44 ꢀ 0.62 12.89 ꢀ 3.45
10.59 ꢀ 4.08
Cmax (nmol mLꢁ1
)
4.22 ꢀ 0.97 5.14 ꢀ 1.96 103.76 ꢀ 22.18
3.05 ꢀ 0.59 37.88 ꢀ 23.47 649.94 ꢀ 91.67
AUC(0–t) (nmol mLꢁ1 hꢁ1
MRT(0–t) (h)
)
1.91 ꢀ 0.38 14.67 ꢀ 1.23
1.54 ꢀ 0.28 0.14 ꢀ 0.07
5.37 ꢀ 1.58 2.65 ꢀ 1.63
12.82 ꢀ 1.39
0.006 ꢀ 0.002
0.091 ꢀ 0.022
CLz (L hꢁ1 kgꢁ1
)
Fig. 5 Pharmacokinetic curves of Taxol® and the PTX-SA LPs after a single
intravenous administration of the equivalent PTX dose of 4 mg kgꢁ1. Data
are presented as mean ꢀ SD (n ¼ 3).
Vz (L kgꢁ1
)
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Fig. 6 In vivo biodistributions of Taxol® and PTX-SA LPs in the heart (A), spleen (B), liver (C), lungs (D), kidneys (E) and tumors (F) in 4T1 tumor-
bearing BALB/c mice. Data are presented as mean ꢀ SD (n ¼ 3).
signicant variations in the hematological parameters in any of
the groups. The H&E staining results are shown in Fig. 8. No
noticeable pathological variations were observed for the H&E
staining of vital organs. The tumor segment results revealed
that the PTX-SA LPs could induce necrosis or apoptosis of tumor
cells; furthermore, the effect of the high-dose group was more
pronounced. All these results suggest that the PTX-SA LPs are
more effective formulations in comparison to Taxol®, with
negligible toxicity to major organs and tissues.
3.2 Synthesis of weak-acid PTX derivatives
3.2.1 Synthesis of PTX-SA or PTX-GA. PTX (0.85 g, 1 mmol),
succinic anhydride (0.1 g, 1 mmol) or glutaric anhydride (0.12 g,
1 mmol) and DMAP (0.024 g, 0.2 mmol) were dissolved in 30 mL
anhydrous dichloromethane. The reaction mixture was stirred
for 12 h at room temperature under nitrogen atmosphere, and
the progress of the reaction was monitored by thin-layer chro-
matography (TLC). Aer the reaction, the solution was placed in
a separating funnel, washed twice with 0.5 M HCl and once with
water, and was evaporated to dryness to obtain a crude product.
The crude product was puried by preparative high perfor-
mance liquid chromatography (pre-HPLC) with the following
solvent ratio: 80% acetonitrile and 20% water, with 0.1% formic
acid added (v/v). The ow rate was 5.0 mL minꢁ1. The UV
detector was maintained at 227 nm. The nal product was
a white solid (85% yield). The target products were conrmed by
mass spectrometry (Agilent 1100 Series LC/MSD Trap) and
3. Experimental section
3.1 Materials and reagents
Paclitaxel (PTX) was purchased from Jingzhu Bio-technology Co.
Ltd. (Nanjing, China). Succinic anhydride (SA), glutaric anhy-
dride (GA), trans-2-butene-1,4-dicarboxylic acid (DA), N-(3-
dimethylaminopropyl)-N-ethyl-carbodiimide
hydrochloride
1
nuclear magnetic resonance spectroscopy (400 MHz H NMR,
(EDCI) and 4-dimethylaminopyrideine (DMAP) were provided
by Aladdin Industrial Corporation (Shanghai, China). 1,2-Dis-
tearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (for
injection) (Chol) and 2-distearoyl-sn-glycero-3-phosphoethanol-
amine-N-methyl (polyethylene glycol)-2000 (DSPE-PEG2000)
were purchased from Shanghai Advanced Vehicle Technology
Pharmaceutical Ltd. (Shanghai, China). Sepharose CL-4B gel
was purchased from Solarbio Corporation. Roswell Park
Memorial Institute (RPMI-1640) and trypsin were obtained from
Gibico (Beijing, China). Fetal bovine serum (FBS) was obtained
from Foundation (Beijing, China), and MTT was purchased
from Meilun Biotech Co. Ltd. (Dalian China). 96-well plates
were supplied by NEST Biotechnology (Jiangsu, China). All other
materials were of analytically pure grade.
Bruker AV-400). The assignments of the NMR peaks and MS
were analyzed as follows:
PTX-SA. 1H NMR (400 MHz, chloroform-d) d 8.07 (2H, Ar–H),
7.68 (2H, Ar–H), 7.54 (1H, Ar–H), 7.44 (3H, Ar–H), 7.32 (7H, Ar–
H), 7.01 (1H, –NH–), 6.22 (1H, 10-H), 6.17 (1H, 13-H), 5.92 (1H,
30-H), 5.61 (1H, 2-H), 5.46 (1H, 20-H), 4.89 (1H, 5-H), 4.36 (1H, 7-
H), 4.23 (1H, 20a-H), 4.14 (1H, 20b-H), 3.72 (1H, 3-H), 2.61–2.52
(4H, –OCCH2CH2–CO–), 2.52 (1H, 6a-H), 2.37 (3H, 4-COCH3),
2.31 (2H, 14-H), 2.14 (3H, 10-COCH3), 1.84 (3H, 18-CH3), 1.84
(1H, 6b-H), 1.61 (3H, 19-CH3), 1.15 (3H, 17-CH3), 1.06 (3H, 16-
CH3). MS (ESI) m/z for C51H54NO17 [M ꢁ H]ꢁ: 952.3394.
PTX-GA. 1H NMR (400 MHz, chloroform-d) d 8.14 (2H, Ar–H),
7.73 (2H, Ar–H), 7.61 (1H, Ar–H), 7.52 (3H, Ar–H), 7.38 (7H, Ar–
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Fig. 7 In vivo anti-cancer efficacy of PTX-SA LPs. (A) Therapeutic schedule for PTX-SA LPs. (B) Tumor growth curves and (C) body weight
changing curves post-injection of Taxol® and PTX-SA LPs (n ¼ 5). (D) Tumor burden rate (n ¼ 5) and (E) hematological and blood biochemical
parameters of different formulations after treatment (the units for AST, ALT, BUN and CR are U Lꢁ1, U Lꢁ1, mmol Lꢁ1 and mmol Lꢁ1, respectively,
n ¼ 3). Data are presented as mean ꢀ SD. *P < 0.05, **P < 0.01, ***P < 0.001.
H), 7.20 (1H, –NH–), 6.30 (s, 1H, 10-H), 6.26 (1H, 13-H), 6.01 (1H, –OCCH2CH2CH2–CO–), 1.66 (3H, 19-CH3), 1.21 (3H, 17-CH3),
30-H), 5.69 (1H, 2-H), 5.49 (1H, 20-H), 4.97 (1H, 5-H), 4.44 (1H, 7- 1.13 (3H, 16-CH3). MS (ESI) m/z for C52H56NO17 [M ꢁ H]ꢁ: 967.1.
H), 4.29 (1H, 20a-H), 4.20 (1H, 20b-H), 3.82 (1H, 3-H), 2.56 (1H,
3.2.2 Synthesis of PTX-DA. PTX (0.85 g, 1 mmol), trans-2-
6a-H), 2.47 (3H, 4-COCH3), 2.42 (2H, –OCCH2CH2CH2–CO–), butene-1,4-dicarboxylic acid (0.22 g, 1 mmol) and DMAP (0.06 g,
2.38 (2H, –OCCH2CH2CH2–CO–), 2.31 (2H, 14-H), 2.22 (3H, 10- 0.2 mmol) were dissolved in 30 mL anhydrous dichloromethane
COCH3), 1.94 (3H, 18-CH3), 1.86 (1H, 6b-H), 1.86 (2H, and stirred for 40 min at 0 ꢂC; then, EDCI (0.38 g, 2 mmol) was
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Fig. 8 The H&E staining histological images of the major organs and tumors. The scale bar represents 100 mm.
added, and the mixture was continuously stirred for 1 h. Next, vesicles with uniform sizes. To establish the ion gradient
the reaction mixture was returned to room temperature for 2 h between the intra-liposomal and extra-liposomal phases, the
under nitrogen atmosphere, and the progress of the reaction samples of extruded liposomes were passed through a Sephar-
was monitored by thin-layer chromatography (TLC). Aer the ose CL-4B gel column pre-equilibrated with 120 mM sodium
reaction, the solution was handled with the same operations sulfate.
described previously to obtain a crude product. Then, the crude
The blank non-gradient liposomes were prepared by a nearly
product was puried by column chromatography to obtain identical process to that used to prepare the blank gradient
a white solid (74% yield). The target product was conrmed by liposomes except that the hydration medium was replaced by
mass spectrometry (Agilent 1100 Series LC/MSD Trap) and 120 mM sodium sulfate and the process of exchanging the extra-
1
nuclear magnetic resonance spectroscopy (400 MHz H NMR, liposomal aqueous phase was unnecessary.
Bruker AV-400). The assignment of the NMR peaks and MS was
analyzed as follows:
PTX-SA, PTX-GA, or PTX-DA was dissolved in DMSO at
a concentration of 5 mg mLꢁ1. Then, the drug solution was added
PTX-DA. 1H NMR (400 MHz, chloroform-d) d 8.06 (2H, Ar–H), to the blank gradient or non-gradient liposomeꢂs (5 mg mLꢁ1 for
7.66 (2H, Ar–H), 7.53 (1H, Ar–H), 7.44 (3H, Ar–H), 7.33 (7H, Ar– lipids) dropwise with continuous stirring at 65 C and incubated
H), 6.95 (1H, –NH–), 6.22 (1H, 10-H), 6.17 (1H, 13-H), 5.91 (1H, for 30 min (the nal drug-to-lipid ratio was 1/10, w/w). Next, the
30-H), 5.61 (1H, 2-H), 5.56–5.51 (2H, –CH]CH–), 5.44 (1H, 20-H), liposomes were quenched immediately for 15 min in an ice bath.
4.89 (1H, 5-H), 4.36 (1H, 7-H), 4.23 (1H, 20a-H), 4.13 (1H, 20b- The residual DMSO and un-encapsulated drug were removed by
H), 3.74 (1H, 3-H), 3.10–2.94 (4H, –OCCH2CH]CHCH2–CO–), dialysis or by passing through a Sepharose CL-4B gel column
2.48 (1H, 6a-H), 2.37 (3H, 4-COCH3), 2.26 (2H, 14-H), 2.15 (3H, equilibrated with 120 mM sodium sulfate.
10-COCH3), 1.84 (3H, 18-CH3), 1.81 (1H, 6b-H), 1.61 (3H, 19-
CH3), 1.15 (3H, 17-CH3), 1.06 (3H, 16-CH3). MS (ESI) m/z for
C
53H56NO17 [M ꢁ H]ꢁ: 978.3571.
3.4 Characterization of liposomes
The particle sizes and polydispersity indices (PDIs) of PTX-SA,
PTX-GA, and the PTX-DA LPs were measured by dynamic light
3.3 Preparation of liposomes and drug loading
Blank gradient liposomes were prepared by the established scattering (DLS) using a Malvern laser particle size analyzer
thin-lm hydration approach. DSPC, cholesterol and DSPE- (Nano ZS, Malvern, UK).
PEG2000 were dissolved in chloroform at a weight ratio of
Gel ltration was applied to determine the encapsulation effi-
68.1 : ꢂ22.2 : 1.2. This solution was dried by a rotary evaporator ciency (EE). Briey, 400 mL of liposome samples were loaded on
at 40 C until a thin lm was formed. Then, 120 mM calcium a 120 mM sodium sulfate pre-saturated Sepharose CL-4B gel
acetate solution was added to the lm, which was hydrated at column to remove the unencapsulated drug, and methanol was
65 ꢂC for 30 min to obtain multilamellar vesicles (MLVs). used as a demulsier. The concentration of drugs was determined
Subsequently, the MLVs were extruded successively through by HPLC on a reverse ODS XB-C18 column (4.6 mm ꢃ 150 mm, 5
a series of polycarbonate lters with pore sizes of 400 nm, mm) with a solvent ratio of 50% ACN and 50% water with 0.1%
200 nm and 100 nm at 65 C under the protection of nitrogen phosphoric acid added (v/v), at a ow rate of 1.0 mL minꢁ1. The UV
ꢂ
using a high-pressure extrusion device in order to obtain small detector was maintained at 227 nm. The encapsulation efficiency
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(EE) of the drugs was calculated according to the following shaker to dissolve the formazan completely. The absorbance
equation:
value of each hole was measured at 570 nm in a microplate
reader. The half maximal inhibitory concentration (IC50) was
obtained by non-linear regression analysis.
EE (%) ¼ C2/C1 ꢃ 100%,
where C2 represents the quantity of drug encapsulated in the
liposomes and C1 is the total quantity of the drug.
3.9 Animals
The morphology of the PTX-SA LPs was observed via trans-
mission electron microscope (TEM, JEM2100, JEOL, Japan). The
samples were prepared by dropping 10 mL diluted PTX-SA LPs
on a carbon-coated copper grid for 30 s and drying the grid with
lter paper. Aerwards, 0.2% uranyl acetate (10 mL) was drop-
ped and retained for 30 s.
Sprague-Dawley (SD) rats (male, 200–230 g) and BALB/c mice
(female, 18–22 g) were provided by the Laboratory Animal
Center of Shenyang Pharmaceutical University. All animal
procedures were performed in accordance with the Guidelines
for Care and Use of Laboratory Animals of Shenyang Pharma-
ceutical University, and the experiments were approved by the
Animal Ethics Committee of Shenyang Pharmaceutical
University.
3.5 Physical stability
In order to evaluate the long-term stability of the liposomes,
PTX-SA LPs (0.5 mg mLꢁ1 for PTX-SA) were stored at 4 C for 1
ꢂ
3.10 In vivo pharmacokinetic study
month. The particle size, PDI and zeta potential were measured
at pre-determined intervals.
Male SD rats were stochastically assigned to two groups (n ¼ 3
per group). Prior to the experiments, the rats were fasted for
12 h with free access to water. Taxol® and PTX-SA LPs with an
equivalent dosage of PTX (4 mg kgꢁ1) were administrated via
tail vein. At the predetermined time points, blood samples were
collected and then centrifuged to obtain the plasma. The ob-
tained plasma samples were deposited at ꢁ20 ꢂC until deter-
mination. The concentrations of PTX-SA and PTX were
measured by UPLC-MS-MS (Waters Corp, Milford, MA, USA),
and the pharmacokinetic parameters were calculated using DAS
2.0 soware in the meantime.
Additionally, l mL PTX-SA LPs (0.5 mg mLꢁ1 for PTX-SA) were
incubated with 9 mL PBS (pH 7.4) containing 10% FBS under
ꢂ
a shaking rate of 100 rpm at 37 C for 48 h. The size and PDI
were measured at given time intervals as well.
3.6 In vitro drug release
The in vitro drug release of the PTX-SA LPs was investigated by
ꢂ
dialysis at 37 C. PTX-SA LPs were added to a dialysis bag and
suspended in 30 mL phosphate buffer saline (PBS, pH 7.4)
containing 0.5% Tween 80 (v/v). At predetermined time points,
500 mL samples were taken for analysis and equal volumes of
medium were replenished. The concentrations of PTX and PTX-
SA were detected by HPLC as mentioned in 3.4.
3.11 In vivo biodistribution study
4T1 tumor-bearing BALB/c mice were utilized to access the
biodistribution of the PTX-SA LPs. When the tumor volume
reached approximately 300 mm3, Taxol® and PTX-SA LPs at
a dose equivalent to 8 mg kgꢁ1 of PTX were administrated via
tail vein (n ¼ 9 for each group). At 6, 24 and 48 h post injection,
the mice were sacriced and the major organs (heart, liver,
spleen, lung and kidney) and tumors were collected and
3.7 Cell culture
4T1 (breast cancer) cells were cultured in Gibico 1640 medium
with 10% FBS, penicillin (30 mg mLꢁ1) and streptomycin (100 mg
mLꢁ1). RM-1 (prostatic cancer) cells were cultured in Gibico 1640
medium with 10% FBS, penicillin (30 mg mLꢁ1), streptomycin (100
mg mLꢁ1), and glucose (2.5 mg mLꢁ1). All cells were maintained in
a humidied atmosphere of 5% carbon dioxide (CO2) at 37 ꢂC.
ꢂ
deposited at ꢁ20 C until analysis. The concentrations of PTX-
SA and PTX were measured by UPLC-MS-MS (Waters Corp,
Milford, MA, USA).
3.8 Cytotoxicity assay
3.12 In vivo antitumor efficacy study
The cytotoxicities of PTX solution, PTX-SA solution and the PTX-
SA LPs against 4T1 cells and RM-1 cells were determined using Mice bearing 4T1 tumors were utilized to estimate the in vivo
the MTT assay. Briey, cells were seeded in 96-well plates at antitumor activity. Approximately 5 ꢃ 106 of the 4T1 cells in 200
a density of 1000 cells per well and incubated for 24 h. Then, the mL PBS were subcutaneously injected in the right ank of female
culture medium was replaced by 200 mL of fresh medium which BALB/c mice. When the tumor volume reached around 100
contained PTX solution, PTX-SA solution or PTX-SA LPs, mm3, the mice were randomly divided into 4 groups (n ¼ 5). The
respectively. Fresh culture medium without drugs served as animals in each group were administered every three days via
a control (n ¼ 3 for each group). The plates were incubated for tail vein for a total of four times with saline, PTX solution (8 mg
48 or 72 h.
kgꢁ1), PTX-SA LPs (equivalent to 8 mg kgꢁ1 PTX), or PTX-SA LPs
Aer that, at pre-determined time points, 20 mL of the MTT (equivalent to 30 mg kgꢁ1 PTX), respectively. The tumor
solution was added to each well containing cells. Aer cultiva- volumes and the body weights of the mice were monitored every
tion for 4 h, the solution was removed completely and 200 mL of two days. On the eleventh day, all mice were sacriced and their
DMSO was added to each well in order to dissolve the formazan blood was collected for hepatic and renal function analysis.
crystals. Next, the plates were vibrated for 10 min on a mini Meanwhile, the major organs and the tumors were dissected
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and xed in 4% paraformaldehyde for hematoxylin and eosin
(H&E) staining.
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Authorship contribution statement
Jiang Yu: investigation, writing—original dra, visualization,
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Zhonggui He: resources. Guimei Lin: writing—review and
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Wang: conceptualization, methodology, writing—review and
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Acknowledgements
This research was supported by National Science and Tech-
nology Major Projects for Major New Drugs Innovation and
Development (No. 2017ZX09101-001-005, Beijing, China), the
Science and Technology Plan Project of Shenyang (No. 18-400-4-
08, Z17-5-064) and the Career Development Program for Young
and Middle-aged Teachers in Shenyang Pharmaceutical
University.
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