Pluronic-poly[α-(4-aminobutyl)-l-glycolic acid] polymeric micelle-like nanoparticles as carrier for drug delivery

Article abstract of DOI:10.1166/jnn.2014.8666

Pluronic-poly[α-(4-aminobutyl)-l-glycolic acid] (Pluronic-PAGA) with different types of Pluronic, the different molecule weight of PAGA, and the different molar ratios of Pluronic to PAGA were synthesized. These materials were bio-degradable, amphiphilic, could be degraded into non-toxic small molecules and could be used to carry drugs. 5-Fluorouracil (5-Fu) loaded Pluronic-PAGA micellelike nanoparticles (5-Fu loaded P-PAGA NPs) were prepared by a simple self-assembly method, and characterized by dynamic light scattering, transmission electron microscope. The degradation and release characteristics have also been studied in this paper. With the time passing, the 5-Fu loaded P-PAGA NPs degraded into smaller ones with the similar characteristics of the original NPs. Both the types of Pluronic and the molecule weight of the PAGA affected the releasing progresses. It was found that 5-Fu loaded P-PAGA NPs exhibited high growth inhibitory effect on human gastric cancer cells by MTT assay. The cellular uptake of Rhodamine B loaded P-PAGA NPs was higher than free Rhodamine B. This study suggested that the Pluronic-PAGA with acceptable drug entrapment efficiencies, drug loading efficiencies and tunable release profiles could offer an alternative carrier for 5-Fu delivery and have the potential for the delivery of other anti-tumour drug. Copyright

Full text of DOI:10.1166/jnn.2014.8666

Article
Journal of
Nanoscience and Nanotechnology
Vol. 14, 4843–4850, 2014
Copyright © 2014 American Scientific Publishers
All rights reserved
Printed in the United States of America
www.aspbs.com/jnn
Pluronic-Poly[ꢀ-(4-aminobutyl)-l-glycolic acid] Polymeric
Micelle-Like Nanoparticles as Carrier for Drug Delivery
1
ꢀ2
1
1
1ꢀ2
1
Qi Wang , Peifeng Liu , Ying Sun , Heng Wu , Xiaoyu Li ,
1ꢀ∗
2ꢀ∗
Yourong Duan , and Zhirong Zhang
1Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine,
Shanghai 200032, China
2
Key Laboratory of Drug Targeting and Novel Drug Delivery Systems, Ministry of Education,
West China School of Pharmacy, Sichuan University, Chengdu, Sichuan 610041, China
Pluronic-poly[ꢀ-(4-aminobutyl)-l-glycolic acid] (Pluronic-PAGA) with different types of Pluronic, the
different molecule weight of PAGA, and the different molar ratios of Pluronic to PAGA were synthe-
sized. These materials were bio-degradable, amphiphilic, could be degraded into non-toxic small
molecules and could be used to carry drugs. 5-Fluorouracil (5-Fu) loaded Pluronic-PAGA micelle-
like nanoparticles (5-Fu loaded P-PAGA NPs) were prepared by a simple self-assembly method,
and characterized by dynamic light scattering, transmission electron microscope. The degradation
and release characteristics have also been studied in this paper. With the time passing, the 5-Fu
loaded P- PDA eG l Ai v eN r Pe sd db ey g rPa ud be dl isi hn ti on gs mT ae l cl ehr no on l eo sg yw itt oh :t hU en si vi me ri lsa i rt yc ho af r Na ce t ew ri Ss t oi c us t ho f Wt h ea l oe rsi ginal NPs.
Both the types of Plur oI Pn i :c 5a . n1 d0 1t h. e2 2m1 o. 1l e 8c 4u l eO wn :e iSg ah tt ,o 1f 6t h Ae pP rA 2G 0A 1 a6 f f1e 5c t: e2 d8 :t 1h e9 releasing progresses.
It was found that 5-Fu load Ce do pP y- Pr iAg Gh At : AN mP se er ixc ha i bn it Se dc i he i ng th if igc r oPw ut hb l ii ns hh i eb ri t so ry effect on human gastric
cancer cells by MTT assay. The cellular uptake of Rhodamine B loaded P-PAGA NPs was higher
than free Rhodamine B. This study suggested that the Pluronic-PAGA with acceptable drug entrap-
ment efficiencies, drug loading efficiencies and tunable release profiles could offer an alternative
carrier for 5-Fu delivery and have the potential for the delivery of other anti-tumour drug.
Keywords: Pluronic, Drug Delivery, Poly[ꢀ-(4-aminobutyl)-l-glycolic acid], Self-Assembly.
1
. INTRODUCTION
PAGA (poly[ꢀ-(4-aminobutyl)-l-glycolic acid]) has
been found to be promising in gene delivery. These carrier
systems have been devised for the delivery of DNA,
Micelle-like nanoparticles prepared using organic solvent
had already been studied by many research groups.
In aqueous systems, hydrophobic fragments of amphiphilic
molecules form the core of a micelle, while hydrophilic
fragments form the micelle’s corona.
encapsulated by simple mixing with the copolymer.
Polymeric micelles are considered to be more stable
than micelles prepared by conventional detergents and
can extend circulation time to achieve favorable bio-
distribution and lower toxicity of drugs. Towards hetero-
cyclic compounds, Pluronic block copolymers have much
1
–15
2
6–27
improving their bioavailability.
In order to combine the
advantages of both the Polymeric and the PAGA, Pluronic-
PAGA copolymer was synthesized. Pluronic-PAGA which
is biodegradable is a good candidate for drug and gene
delivery. After degradation of PAGA, Pluronic-PAGA can
exhibit smaller Pluronic micelles.
1
6–22
Drug can be
2
3
Fluorouracil
(5-Fluoro-2,4(1H,3H)-pyrimidinedione,
2
4
5-Fu) is an analogue of the naturally occurring pyrimidine,
uracil, with fluorine substitution in the 5 position which
has unusual toxicity, including myelosupression, mucositis
2
5
higher solubilization capacities and are more selective.
2
8
and, rarely, a cerebellar syndrome. It has accrued a long
clinical history as both a palliative and therapeutic treat-
ment modality for a variety of carcinomas, especially those
Pluronic F108 and Pluronic P105 with PEO block, gave
the most stable formulations on account of having the
capacities to form nanoparticles and to provide hydropho-
bic environment to maintain the drug in monomeric form.
2
9
involving the breast, colorectal region and skin. The load-
ing of this hydrophilic drug to a carrier system will increase
its therapeutic efficacy. This is because the controlled
∗
Authors to whom correspondence should be addressed.
J. Nanosci. Nanotechnol. 2014, Vol. 14, No. 7
1533-4880/2014/14/4843/008
doi:10.1166/jnn.2014.8666
4843

Pluronic-PAGA Polymeric Micelle-Like Nanoparticles as Carrier for Drug Delivery
Wang et al.
delivery to the targeted organ could reduce the severe
chemotherapy side effects. To incorporate anti-cancer
agents into nanoparticles different techniques have been
used, with both preformed polymers and monomers being
3
0
used in their manufacture. Because of the hydrophilicity
of the materials, there are still some problems in the for-
mation of nanoparticle. In order to solve these problems
5
-Fluorouracil (5-Fu) loaded Pluronic-PAGA micelle-like
nanoparticles (5-Fu-P-PAGA NPs) had been prepared in
this article.
In addition of that, common ways to encapsulate drugs
with poor solubility are using certain clinically accept-
able excipients and/or some other organic solvents such
as THF and DMSO, and/or certain surfactants in drug
ꢁ
Figure 1. Synthetic scheme of N -cbz-PAGA.
a white crystal (s)-6-[[(Phenylmethoxy)carbony]amino]-
2-hydroxyhexanoic acid (PCAHA). The white crystal
PCAHA was added into an oven-dried reaction tube and
nitrogen-purged three times. High vacuum was applied at
50 C. After 5 days of reaction, the crude polymer was
3
1
formulations. However, the administration of many co-
solvents or surfactants may cause toxicity or other unde-
3
2
sirable side effects. In our studies, 5-Fu-P-PAGA NPs
could be prepared without organic solvents.
ꢁ
1
Then, the parameters that influence the amount of 5-Fu
loading to the Pluronic-PAGA NPs, both on the sur-
face and in the polymeric network, are studied: these
are, specifically, preparation methods, the drug/copolymer
ratio, the types of Pluronic and the Pluronic/PAGA ratio.
Some factors which would determinate the release profiles
in vitro had also been investigated, including the type of
Pluromic, hte Pluronic/PAGA ratio and the type of drug
incorporation.
dissolved in chloroform and purified by excess methanol
to give a white powder N -cbz-PAGA.
ꢁ
2.2.2. Synthesis of Pluronic-PAGA
Pluronic-PAGA with different Pluronic/PAGA ratio was
prepared. Figure 2 shows the reaction scheme for the syn-
ꢁ
thesis of Pluronic-PAGA. Briefly, N -cbz-PAGA and NHS
ꢁ
in CH Cl at 0 C were treated with DCC. The mixture
2
2
Delivered by Publishing Technology t ow: aUs nsi tvi er r resd it ya to rf oNo em wt eS mo pu et hr a Wt u rae lef so r 16 h, and then, the
The main aim of this research is to further improve the
IP: 5.101.221.184 On: Sat,s 1o 6l i dA pD rC2 U0 1 b6 y 1p r5 o: d2 u8 c: 1t 9w as filtered off and washed with
co-carrying of drug and RNA of Pluronic/PAGA and pro-
Copyright: American Scientific Publishers
CH Cl . Pluronic dissolved in CH Cl was added to the
2
2
2
2
mote its clinical application.
solution. The mixture was stirred at room temperature for
6 h. After concentrating in vacuo, Pluronic-PAGA-cbz-
1
ꢁ
2
. MATERIALS AND METHODS
N was purified by excess ether. Deprotection of cbz group
ꢁ
2
.1. Materials
from Pluronic-PAGA-cbz-N was done by hydrobromic
acid/acetic acid. The precipitate was obtained by pouring
the reaction solution into an excess of chilled ether. The
copolymer Pluronic-PAGA was collected by vacuum fil-
tration and dried in vacuo at room temperature.
Samples of Pluronic P105 and Pluronic F108 were
kindly supplied by BASF China Ltd. (Shanghai, China).
5
-fluorouracil (5-Fu) was purchased from Shanghai
Sanjiang Bio-Engineering Co. Ltd. (Shanghai, China).
ꢁ
N -cbz-lysine and N-Hydroxy succinimide (NHS) were
The average molecular weights of Pluronic-PAGA were
12000 to 30000 and the molar ratio of Pluronic to PCAHA
were 1:25, 1:47 and 1:62, respectively. All the Pluroni-
purchased from Acros and used without further purifica-
ꢀ
tion. N,N -Dicyclohexycarbodiimide (DCC) was manufac-
ꢁ
tured from Sinopharm Chemical Reagent Co., Ltd. All
these materials were used without further purification. All
the other reagents were analytical grade.
PAGA samples stored at 4 C.
2.2.3. Preparation of 5-Fu Loaded Pluronic-PAGA
Micelle-Like Nanoparticles ꢄ5-Fu Loaded
2
.2. Methods
P-PAGA NPsꢅ
2
.2.1. Synthesis of Polyꢂꢀ-(4-aminobutyl)-L-glycolic
Pluronic-PAGA was used to prepare 5-Fu loaded P-PAGA
NPs. Both the Pluronic-PAGA and 5-Fu were perfectly
dried under vacuum before use. There are two methods to
prepare 5-Fu loaded P-PAGA NPs by using the different
solvent.
The first method had used organic solvent. A certain
amount of Pluronic-PAGA and 5-Fu were dissolved in
DMF, and then the solution was added drop-wise to dis-
tilled water under gentle stirring. This system was dialyzed
against distilled water for 2 h using a dialysis membrane
ꢁ
acidꢃ ꢄN -cbz-PAGAꢅ
Figure 1 shows the reaction scheme for the synthesis
ꢁ
ꢁ
of N -cbz-PAGA. N -cbz-PAGA was first synthesized by
Lim.33 Briefly, a solution of NaNO in water was added
2
ꢁ
during 3 h to ice-cooled and stirred solution of N -cbz-
lysine in 2NH SO /acetonitrile (1:1). The mixture was
stirred for an additional 2 h. After concentrating in vacuo,
the resulting clear solution extracted with ether and was
then recrystallized from ether/petroleum ether to give
2
4
4
844
J. Nanosci. Nanotechnol. 14, 4843–4850, 2014

Wang et al.
Pluronic-PAGA Polymeric Micelle-Like Nanoparticles as Carrier for Drug Delivery
Figure 2. Synthesis route for Pluronic-PAGA copolymer.
to remove organic solvent. The self-assembly of 5-Fu and
Pluronic-PAGA was shown in Figure 3.
suspended in bi-distilled water and analyzed in duplicate
with five readings per sample.
The second method had used the water as the only sol-
vent. 5-Fu mixed with Pluronic-PAGA was dissolved in
water at room temperature (5-Fu: Pluronic-PAGA = 1:2,
Each nanoparticles was extruded through a membrane of
pore size 3 k. The medium removed from centrifuge was
carefully collected and the free drug amount was obtained
from the concentration of the drug in the medium. The
content of 5-Fu was measured using UV-Visible Spec-
trometer (Shimadzu 1650 PC) at the maximum absorbance
wavelength of 5-Fu (266 nm).
1
:5, 1:10 w:w) under magnetic stirring. Then the 5-Fu
loaded P-PAGA NPs formed. Figure 3 had showed the
self-assembly of 5-Fu and Pluronic-PAGA.
The unincorporated drug is removed during the ultrafil-
tration by passing thro Du ge hl i v3 e kr e Cd e bn tyr i Pf u ug ba ll i sfi hl ti ne rg u Tn ei t cs h( Mn oi ll o- gy to: University of New South Wales
Theoretical drug content (%)
IP: 5.101.221.184 On: Sat, 16 Apr 2016 15:28:19
lipore). Mannitol 10% (w/v) was added to the nanoparticle
Copyright: American Scientific Publishers
suspension and lyophilized, and kept in desiccators. All of
the samples had been listed in Table I.
Weight of copolymer and drug used
= (Weight of drug used in formulation
/
2
A
(
.2.4. Characterization of 5-Fu Loaded P-PAGA NPs
dynamic light scattering particle size analyzer
NICOMP 380 ZLS Zeta Potential/Particle Sizer, PSS
in the formulation)×100%
Entrapment efficiency (%)
(Weight of drug in the nanoparticles
(1)
=
Nicomp, Santa Barbara, CA, USA) was used to mea-
sure the final nanoparticle size distributions of the all
samples. All measurements were performed at 25 C and
/
Weight of drug used in the formation)×100% (2)
ꢁ
ꢁ
the measurement angle was 90 . Each nanoparticles was
Drug loading efficiency (%)
=
(Weight of drug in the nanoparticles/
Weight of copolymer and drug
in the formation)×100%
(3)
According to the result of the size and drug loading effi-
ciency Pluronic105-PAGA-c, Drug:copolymer 1:2 (w:w)
was chosen to doing the morphological examination
which was performed by transmission electron microscope
(TEM) (HITACHI H-7650, Japan). The sample was pre-
pared by wetting a carbon-coated grid with a small drop
of the dilute nanoparticle solution. It was dried for several
minutes at room temperature before analysis.
2.2.5. In Vitro Degradation
The Pluronic105/PAGA (Drug:copolymer 1:2 (w:w))
micelle-like nanoparticles had been chosen as the sample
Figure 3. Schematic illustration of self-assembly of 5-Fu and Pluronic-
PAGA.
J. Nanosci. Nanotechnol. 14, 4843–4850, 2014
4845

Pluronic-PAGA Polymeric Micelle-Like Nanoparticles as Carrier for Drug Delivery
Wang et al.
Table I. Characteristics of Pluronic-PAGA nanoparticles.
Pluronic:
PCAHA (M:M)
Drug: copolymer
(w:w)
Sample
number
Theoretical drug
content (%)
Entrapment
efficiency (%)
Drug loading
efficiency (%)
Copolymer
Pluronic P105-PAGA-a
1:25
1:47
1:62
1:25
1:47
1:2
1
2
33ꢆ78
33ꢆ43
26ꢆ54
53ꢆ72
8ꢆ97
17ꢆ96
∗
∗
∗
1:5
3
4
16ꢆ71
16ꢆ79
28ꢆ45
56ꢆ41
4ꢆ75
9ꢆ47
∗
∗
∗
1:10
5
9ꢆ86
26ꢆ88
2ꢆ65
Pluronic P105-PAGA-b
Pluronic P105-PAGA-c
Pluronic F108-PAGA-a
Pluronic F108-PAGA-b
Pluronic F108-PAGA-c
1:2
6
7
32ꢆ92
33ꢆ23
39ꢆ5
66ꢆ59
13
22ꢆ13
∗
∗
∗
1:5
8
9
16ꢆ79
16ꢆ92
39ꢆ2
61ꢆ77
6ꢆ58
10ꢆ45
∗
∗
∗
1:10
10
10ꢆ17
35ꢆ75
3ꢆ63
1:2
11
35ꢆ55
33ꢆ01
45ꢆ34
81ꢆ55
16ꢆ12
23ꢆ61
∗
∗
∗
12
1:5
13
16ꢆ9
16ꢆ81
41ꢆ58
64ꢆ36
7ꢆ03
10ꢆ82
∗
∗
∗
14
1:10
15
9ꢆ99
35ꢆ68
3ꢆ56
1:2
16
35ꢆ01
32ꢆ87
26ꢆ63
54ꢆ07
9ꢆ31
17ꢆ77
∗
∗
∗
17
1:5
18
16ꢆ86
16ꢆ69
32ꢆ71
5ꢆ51
9ꢆ50
∗
∗
∗
19
56ꢆ9
1:10
20
10ꢆ26
13ꢆ5
1ꢆ39
1:2
21
32ꢆ93
33ꢆ17
26ꢆ01
52ꢆ81
8ꢆ56
17ꢆ52
∗
∗
∗
22
1:5
23
16ꢆ97
16ꢆ75
32ꢆ45
56ꢆ82
5ꢆ51
9ꢆ52
∗
∗
∗
24
1:10
25
9ꢆ89
5ꢆ27
0ꢆ52
Delivered by Publishing Technology to: University of New South Wales
1:62 IP: 5.101.221 .11: 28 4 On: Sat, 12 66 Apr 2016 13 35 ꢆ :4 27 8:19
31ꢆ45
63ꢆ06
10ꢆ53
20ꢆ69
Copyright: American Sci e2 7n tific Publis 3h 2eꢆ7r 1s ∗
∗
∗
∗
1:5
28
16ꢆ71
16ꢆ72
34ꢆ83
59ꢆ75
5ꢆ82
9ꢆ99
∗
∗
29
1:10
30
10ꢆ16
31ꢆ59
3ꢆ21
∗
Note: “ ” refers to: nanoparticles prapared with DMF.
for degradation study. The 5-Fu loaded polymer aggregates
were added into a glass bottle with phosphate-buffered
at the maximum absorbance wavelength of 5-Fu (266 nm).
All release measurements were carried out in duplicate for
each material and the average values were plotted.
saline (PBS) aqueous solution. The glass bottle was then
ꢁ
agitated in a horizontal shaker maintained at 37
C
for 2 h. The Pluronic105/PAGA ( Drug:copolymer 1:2
w:w)) micelle-like nanoparticles and the sample which
had degraded for 2 h had both been examined by dynamic
light scattering particle size analyzer to understand the
morphologies changes with time.
2.2.7. Cell Culture
(
SGC-7901 cell line (a human gastric cancer cell line)
was provided by Shanghai Institutes for Biological Sci-
ences (Shanghai, China) and was cultured in RPMI
1640 (GIBCO, USA) supplemented with 10% calf serum
(
Minhai, Gansu, China) and 100 IU/mL penicillin and
ꢁ
2
.2.6. Drug Release In Vitro
A certain amount of sample was added into a bag filter
exclusion molecular weight 20000) placed in a glass bot-
100 ꢇg/mL streptomycin. Cells were maintained at 37 C
in a humidified atmosphere containing 5% CO .
2
(
tle with phosphate-buffered saline (PBS) aqueous solution.
Free 5-Fu had been used as the control. The glass bot-
2.2.8. MTT Assay
The cytotoxicity of 5-Fu loaded P-PAGA NPs against to
SGC-7901 cells was determined by MTT assay. Sam-
ple 12 (Table I) has been chosen to complete this assay.
The cells were seeded in 96-well culture plates at a den-
tle was then agitated in a horizontal shaker maintained at
ꢁ
3
7 C for 7 days for the drug release study. At prede-
termined intervals, 1 mL samples were removed from the
release medium and PBS was added with the same volume
and temperature. The amount of the drug released from the
nanoparticles was monitored by UV-Visible spectrometer
4
sity of 1 × 10 cells/well. And the MTT assays were
performed at 24 h with a series concentrations of 5-Fu
(5, 10, 20, 40, and 80 ꢇg/mL 5-Fu, respectively) and 5-Fu
4
846
J. Nanosci. Nanotechnol. 14, 4843–4850, 2014

Wang et al.
Pluronic-PAGA Polymeric Micelle-Like Nanoparticles as Carrier for Drug Delivery
loaded P-PAGA NPs (5, 10, 20, 40, and 80 ꢇg/mL 5-Fu,
respectively with 10, 20, 40, 80, and 160 ꢇg/mL P-PAGA)
and blank P-PAGA NPs (10, 20, 40, 80, and 160 ꢇg/mL
P-PAGA respectively). A mixture of 20 ꢇL of MTT solu-
tion (5 mg/mL in PBS, pH 7.4) was added to each well.
In the absence of light, samples were incubated for 4 h
and the precipitates were resuspended by adding 100 ꢇL
of DMSO to each well in order to ensure solubilization
of the formazan crystals. The absorbance was measured
on a microplate reader (Bio-Rad Model 550, USA) at
5
70 nm. The growth inhibition ratio (GIR) was calculated
according to the following equation and the 50% inhibitory
concentration (IC50) of 5-Fu loaded P-PAGA NPs was
evaluated as well.
Figure 4. The diameters of 5-Fu loaded P-PAGA nanoparticles.
((a) P105-PAGA-a; (b) P105-PAGA-b; (c) P105-PAGA-c; (d) F108-
PAGA-a; (e) F108-PAGA-b; (f) F108-PAGA-c). Data as mean ± S.D.
ꢄn = 3ꢅ.
GIR = (1−OD of tested sample/OD of negative control)
process of micelle-like nanoparticle formation, on which
there are lots of factors that may have an impact.
×100
Such self-assembly nanoparticles have not yet been pre-
pared without the use of organic solvent, such as using
only water. The technique developed in our present work
not only is simple, but also can exclude the influence of the
organic solvents. However, we should concede that DMF
produces better nanoparticles than water. But using a novel
self-assembly method, the evaporation or dialysis time was
reduced by eliminating organic solvents from the process.
2
.2.9. Uptake Study of P-PAGA NPs by SGC-7901 Cells
Rhodamine B (Ex = 570 nm, Em = 595 nm) loaded
P-PAGA NPs was prepared by the method without organic
solvents (Rhodamine B:Pluronic-PAGA = 1:10, w:w).
2
The SGC-7901 cells were seeded in 25 cm flasks (1×
6
1
0 cells/flask) and then exposed to free Rhodamine B
or Rhodamine B loaded P-PAGA NPs at a final dose of
0 ꢇg/mL during the exponential growth phase. After they
were incubated with SGC-7901 cells in triplicate for 0.5,
1
Delivered by Publishing Technology to: University of New South Wales
.2. Characterization of Nanoparticles
3
2
, or 4 h, respectively, 2 mL of PBI PS :( p5 H. 1 07 1. 4. )2 w2 1e .r 1e 8u 4s e Od nt o: Sat, 16 Apr 2016 15:28:19
In order to analyze the influence factors on the physico-
wash cells in order to remove extracell Cu l oa rp yd rr iug ghs t. : 5A ꢇm g e/ mr i cL an Scientific Publishers
chemical characteristics of the 5-Fu-P-PAGA NPs, as well
of DAPI (Ex = 340 nm, Em = 488 nm) was added for
another 10 min. After the staining solution was removed,
cells were rinsed with PBS (pH 7.4) three times, fixed with
as on the size, encapsulation capacity and controlled release
properties, and to choose the optimum conditions, the char-
acterization of all the samples (Table I) were determined.
Figure 4 shows the diameters of 5-Fu-P-PAGA NPs.
The measurement reveals that all samples had small diam-
eters which were from 190 to 260 nm. As we known,
the transport and accumulation efficiency of NPs into the
tumor interstitium are limited by their ability to penetrate
4% paraformaldehyde for 5 min. The cells were imaged
using Fluorescence microscopy.
3
3
. RESULTS AND DISCUSSION
.1. Preparation of 5-Fu Loaded P/PAGA NP
3
4
As the steps described before, 5-Fu loaded P-PAGA NPs
were prepared. Pluronic-PAGA was used as the main com-
ponent for the preparation of nanoparticles (self-assembled
aggregates). In the present work, we have prepared
biodegradable nanoparticles using a solvent diffusion tech-
nique that was conveniently adapted for the encapsulation
of hydrosoluble macromolecules. Since 5-Fu molecules
contain heterocyclic group, it seems to be suitable for the
preparation of nanoparticles with this drug into Pluronic.
All kinds of nanoparticles had been showed in Table I.
Actually, the 5-Fu was incorporated into Pluronic-PAGA
nanoparticles through a simple self-assembly procedure,
as shown in Figure 4. When the drug and Pluronic-PAGA
were dissolved in water, micellar solution would be formed
and hydrophobic 5-Fu would be spontaneously incorpo-
rated into hydrophobic block of Pluronic copolymer. It is
essentially a reversible aggregation process induced by
limited aqueous solubility of the Pluronic-PAGA in the
tumor vascular endothelium. Diffusion and accumulation
of NPs are determined by the cutoff size of the tumor vas-
3
5
culature, and this cutoff size varies for different tumors.
Clearly, the average diameter of the micelles increased
with the weight of copolymer. The factors, the ratio of the
Pluronic to PAGA and the types of Pluronic, only had little
effect on diameter. The results informed that the ratio of
drug/copolymer was the most important factor for deciding
the size of the nanoparticles.
Beside the size, other characteristics including the
entrapment efficiency (EE) and the drug loading efficiency
(LE) are listed in Table I. From Table I, we can see that
the entrapment efficiency and the drug loading efficiency
of the NPs prepared without using organic solvent were
relatively lower. For sample 12, the EE and the LE of
the NPs could reach 81.55% and 23.61%, respectively.
That was relatively higher than sample 11, the NPs pre-
pared without using organic solvent.
J. Nanosci. Nanotechnol. 14, 4843–4850, 2014
4847

Pluronic-PAGA Polymeric Micelle-Like Nanoparticles as Carrier for Drug Delivery
Wang et al.
because of the EPR effect, because of these advantages,
the Pluronic/PAGA block copolymer was suitable to deliv-
ery drug to tumor.
3.4. Drug Release In Vitro
Achieving sink conditions on the receiver side of the mem-
brane depends on a flow rate that is sufficient to remove
all permeated drug. The PBS was used as it simulates the
condition of the human body and it mediates any fluc-
+
−
tuations from the H and OH ions released during the
degradation process.
The in vitro release profile of 5-Fu loaded P-PAGA NPs
(
3
Sample 2, 7, 12, 17, 22 and 27, Table I), obtained at
7 C, can be seen in Figure 7. The 5-Fu of control group
ꢁ
Figure 5. Transmission electron microscope photograph of 5-Fu
loaded Pluronic P105-PAGA (drug:copolymer = 1:2 (w:w)) micelle-like
nanoparticles.
had released about 90% after only 4 h. All 5-Fu loaded
P-PAGA NPs showed sustained release ability compared to
free 5-Fu. In neutral surroundings (pH 7.4), after 30 min,
2
6
0–40% of 5-Fu was released from the NPs, and after 8 h,
5–80% of 5-Fu was released. The initial burst effect of
The morphology of the 5-Fu loaded P-PAGA nanoparti-
cles was investigated with TEM. Figure 5 shows the TEM
images of 5-Fu loaded P-PAGA NPs. The representative
image shows that -Fu loaded P-PAGA NPs with diameters
in the range of 190–210 nm self-assemble into approxi-
mately spherical micelle-like nanoparticles in water.
the drug was probably caused by the large specific surface
area of the NPs. Some of the 5-Fu was adsorbed on the
surface of the NPs.
Drug release from a copolymer was controlled by
two factors: the diffusivity of the drug and the degrada-
3
6
tion of the copolymer. As we discussed, the first burst
release probably was due to the fast diffusion of the
drug accumulated. The last complete release of 5-Fu from
Pluronic-PAGA copolymer correlated well with the rapid
3
.3. Degradation In Vitro
The changes of the size in degradation progress had
been shown in Figures 6(A) and (B). The Figure 6(A)
showed the particulate dispersions before the degrada-
Delivered by Publishing Technology to: University of New South Wales
IP: 5.101.221.184 On: Sat, 16 Apr 2016 15:28:19
37
Copyright: American S cd i ee gn r tai fd i ca t iPo un bol if s nh ae nr os particles. So according to Siepmann,
tion, the Figure 6(B) showed the particulate dispersions
after the degradation. There was only one single distribu-
tion evidently in Figure 6(A) which sized about 200 nm.
In Figure 6(B), two size peaks were observed, the bigger
one was 200 nm and the smaller one was 20 nm. This
result illustrated that, after degradation, the nanoparticles
had degraded into smaller nanoparticles.
The transform from a bigger particle to some small
ones may successfully make a long circulation in vivo,
and 10–20 nm is the size that previously showed maxi-
mal accumulation and the deepest penetration into tumors
the release profiles between 10% and 80% release were
characterized by fitting the following equation by nonlin-
n
ear regression: M /M = kt , where M and M are the
t
ꢂ
t
ꢂ
absolute cumulative amounts of drug released at time t and
infinite time, respectively, k is the release constant, expo-
nent n describes the kinetic and the release mechanism.
For diffusion–degradation controlled drug release system,
n for spherical particles is usually between 0.43 and 0.85.
When n is close to 0.43, diffusion is the major driv-
ing force, and this is normally called the “Fickian dif-
fusion.” When n is close to 0.85, the drug release is
Figure 6. The change in the size distribution of Pluronic105/PAGA (drug:copolymer 1:2 (w:w)) micelle-like nanoparticles as degradation progressed.
Upper: before degradation; lower: after degradation).
(
4
848
J. Nanosci. Nanotechnol. 14, 4843–4850, 2014

Wang et al.
Pluronic-PAGA Polymeric Micelle-Like Nanoparticles as Carrier for Drug Delivery
Figure 8. MTT assays for the growth inhibition ratio of 5-Fu loaded
P-PAGA NPs in SGC-7901 cell line after 24 h. The values are given as
mean ± S.D. ꢄn = 3ꢅ.
Figure 7. Release profile of 5-Fu from Pluronic-PAGA nanoparticles
with drug:copolymer = 1:2.
mainly controlled by degradation. According to our anal-
ysis, for the majority of 5-Fu released (about 70%) from
Pluronic-PAGA nanoparticles, the release mechanism was
mainly dominated by diffusion, not by the degradation
of PAGA. This is reasonable because the degradation of
PAGA blocks should not significantly affect the release
behavior of drugs from Pluronic-PAGA nanoparticles due
to the relatively slow hydrolytic degradation rate of PAGA
blocks.
Figure 9. Intracellular uptake of P-PAGA NPs. Red, Rhodamine B;
blue, nuclei stained with DAPI. (A) Free Rhodamine B; (B) Rhodamine
B loaded P-PAGA NPs.
As shown in Figure 7, we compared F108-PAGA-c
copolymer with P105- DP Ae lGi v Ae -r ce df o br y5 -PF uu b rl iesl he ai ns eg . TW e ec hn no toe l do gy to: University of New South Wales
that F108-PAGA-c showing less IcPo :n 5t r .o1 l0 o1 v. 2e r2 1t h. 1e 8r 4e l Oe a ns e: Sat, 16 Apr 2016 15:28:19
Copyright: American Scientific Publishers
than P105-PAGA-c. This meant that P105-PAGA-c hav-
3.6. Uptake Study of P-PAGA NPs by SGC-7901 Cells
ing a relatively efficient “core” trapping for this drug.
And also, we compared different copolymers with differ-
ent molecule weight of PAGA for 5-Fu release. Increasing
the relative amount of the PAGA slowed down the over-
all release rate. This probably reflected a longer diffusion
path for the drug. The PAGA with higher molecule weight
may form the better spherical core–shell nanoparticles.
The uptake of the P-PAGA NPs by SGC-7901 cells with
incubation time of 0.5 to 4 h was visualized using fluo-
rescence microscopy. The concentration of P-PAGA NPs
suspension was 100 ꢇg/mL. Effect of incubation time on
the cellular uptake is clearly shown in Figure 9. It shows
stronger fluorescence in the cells with the increase of incu-
bation time from 0.5 to 4 h. Fluorescence was weaker in
control cells which had been exposed to the free Rho-
damine B. P-PAGA NPs had remarkably higher uptake
efficiency than the free drug.
3
.5. MTT Assay
In the cytotoxicity study, the growth inhibition ratio (GIR)
of SGC-7901 cells in the presence of 5-Fu loaded P-PAGA
NPs was evaluated by MTT assays. The cytotoxicity of
treated and untreated cells is determined by the MTT assay
which measures the mitochondrial conversion of MTT to
formazan as detected by the change of optical density at
4. CONCLUSIONS
In summary, Pluronic-PAGA was first reported as a drug
delivery system. Pluronic-PAGA with different types of
the Pluronic, the different length of PAGA, and the
different ratio of Pluronic to PAGA have been synthe-
sized. 5-Fu loaded Pluronic-PAGA micelle-like nanopar-
ticles were prepared by a simple self-assembly method.
It was found that the obtained nanoparticles showed near
spherical shape and the sizes were controllable with the
range of 190–260 nm. Compared the nanoparticles pre-
pared with different ratio of Pluronic and PCAHA, drug
entrapment efficiency of nanoparticles at the ratio of 1:62
was higher than others. Kinetic of degradation had played
an important role in the release process of Pluronic-PAGA
5
70 nm.
The growth inhibition curves of SGC-7901 cells were
dependent on the drug concentration, as shown in Figure 8.
It showed that 5-Fu loaded P-PAGA NPs exhibited signifi-
cantly higher inhibition rates than those of the 5-Fu group
at the same concentration (P < 0ꢆ05). A low evidence
of cytotoxicity was observed for the P-PAGA NPs and
the cell viability of around 100% was observed in the
non-treated groups. Revealing that the cells are much more
sensitive to 5-Fu loaded P-PAGA NPs than the free 5-Fu.
The blank P-PAGA NPs showed a negligible cytoxicity.
J. Nanosci. Nanotechnol. 14, 4843–4850, 2014
4849

Pluronic-PAGA Polymeric Micelle-Like Nanoparticles as Carrier for Drug Delivery
Wang et al.
nanoparticles. Drug release studies in vitro showed that
release rate of 5-Fu from PluronicP105-PAGA nanoparti-
cles was slower than that of PluronicF108-PAGA nanopar-
ticles, and the release mechanism was mainly dominated
by diffusion. The type of Pluronic and the molecule
weight of the PAGA may all have effects on the releasing
progress. We believe the Pluronic-PAGA block copolymer
could offer an alternative carrier for 5-Fu delivery, with
acceptable drug entrapment efficiencies, drug loading effi-
ciencies and a tunable release profile, and have the poten-
tial for the delivery of other anti-tumour drug.
The cytotoxicity and absorption tests with SGC-7901
cells showed that P-PAGA NPs had remarkably higher
uptake efficiency than the free drug. Hence, this novel for-
mulation appears to be a promising delivery system with
improved bioavailability, and has a great potential for clin-
ical application.
11. A. Babu, K. Jeyasubramanian, P. Gunasekaran, and R. Murugesan,
J. Biomed. Nanotechnol. 8, 43 (2012).
1
2. E. P. Werbicka, D. Shcharbin, J. Maly, M. Maly, M. Zaborski,
B. Gabara, P. Ortega, F. J. Mata, R. Gómez, M. A. M. Fernandez,
B. Klajnert, and M. Bryszewska, J. Biomed. Nanotechnol. 8, 57
(
2012).
13. B. Xue, Y. Wang, X. Tang, P. Xie, Y. Wang, F. Luo, C. Wu, and
Z. Qian, J. Biomed. Nanotechnol. 8, 80 (2012).
14. Q. Zhang, N. He, L. Zhang, F. Zhu, Q. Chen, Y. Qin, Z. Zhang,
Q. Zhang, S. Wang, and Q. He, J. Biomed. Nanotechnol. 8, 90
(
2012).
1
1
1
1
5. D. Wehrung and M. O. Oyewumi, J. Biomed. Nanotechnol. 8, 161
(2012).
6. J. Fu, D. Wang, T. Wang, W. Yang, Y. Deng, H. Wang, S. Jin, and
N. He, J. Biomed. Nanotechnol. 6, 725 (2010).
7. W. Yang, J. Fu, T. Wang, and N. He, J. Biomed. Nanotechnol. 5, 591
(
2009).
8. T. Wang, L. Xu, S. Li and N. He, Nanoscale 2, 230 (2010).
19. L. Zhang, Z. Lu, X. Li, Y. Deng, F. Zhang, C. Ma, and N. He, Polym.
Chem. 3, 1958 (2012).
2
0. L. Zhang, Z. Wang, Z. Lu, H. Shen, J. Huang, M. Liu, N. He, and
Z. Zhang, Journal of Materials Chemistry B 1, 749 (2013).
1. L. Zhang, Z. Lu, Y. Bai, T. Wang, Z. Wang, J. Chen, Y. Ding,
F. Yang, Z. Xiao, S. Ju, J. Zhu, and N. He, J. Mater. Chem. B 1, 1289
(2013).
Acknowledgments: This research was supported by the
Biological pharmaceutical and agricultural fields from Sci-
ence and Technology Commission of Shanghai Munici-
pality (No. 114119a3300), the National Natural Science
Foundation of China (No. 81101738) and Medical-
Engineering Joint Funds of Shanghai Jiao Tong University
2
22. T. Wang, Z. Q. Feng, N. Y. He, Z. F. Wang, S. Li, Y. F. Guo, and
L. J. Xu, J. Nanosci. Nanotechnol. 7, 4571 (2007).
23. N. Y. He, T. Wang, L. Jiang, D. X. Wang, Y. Hu, and L. Zhang,
J. Nanosci. Nanotechnol. 10, 5320 (2010).
(No. YG2010MS71 and YG2011MS25).
24. P. F. Liu, L. B. Qin, Q. Wang, Y. Sun, M. J. Zhu, M. Shen, and Y. R.
Duan, Biomaterials 33, 6739 (2012).
2
5. R. Nagarajan and K. Ganesh, J. Colloid Interface Sci. 184, 489
Delivered by Publishing Technology to: University of New South Wales
References and Notes
(1996).
IP: 5.101.221.184 On: Sat, 16 Apr 2016 15:28:19
26. J. L. Grem, Invest. New Drugs 18, 299 (2000).
1
. G. D. Venkatasubbu, S. Ramasamy, V. Ramakrishnan, and J. Kumar,
J. Biomed. Nanotechnol. 7, 759 (2011).
. A. Taheri, R. Dinarvand, F. Atyabi, F. Nouri, F. Ahadi, M. H.
Ghahremani, S. N. Ostad, A. T. Borougeni, and P. Mansoori,
J. Biomed. Nanotechnol. 7, 743 (2011).
Copyright: American Scientific Publishers
27. Y. B. Lim, S. O. Han, H. U. Kong, Y. Lee, J. S. Park, B. Jeong, and
S. W. Kim, Pharm. Res. 17, 811 (2000).
2
28. M. M. Jones, M. A. Basinger, and M. A. Holscher, Anticancer Res.
11, 449 (1991).
3
. G. Thirumurugan, V. S. Veni, S. Ramachandran, J. V. L. N. Sesha-
giri Rao, and M. D. Dhanaraju, J. Biomed. Nanotechnol. 7, 659
29. D. D. Lasic, M. P. Frederik, M. C. Stuart, Y. Barenholz, and T. J.
Mclntosh, FEBS Lett. 312, 255 (1992).
(
2011).
30. P. F. Liu, H. Yu, Y. Sun, M. J. Zhu, and Y. R. Duan, Biomaterials
33, 4403 (2012).
4
5
6
7
8
9
. J. Wang, H. Tan, A. Yu, P. Ling, H. Lou, G. Zhai, and J. Wu,
J. Biomed. Nanotechnol. 7, 696 (2011).
31. I. Brigger, C. Dubernet, and P. Couvreur, Adv. Drug Deliv. Rev.
54, 631 (2002).
32. N. Rapoport, W. G. Pitt, H. Sun, and J. L. Nelson, J. Control Release
91, 85 (2003).
33. Y. B. Lim, Y. H. Choi, and J. S. Park, J. Am. Chem. Soc. 121, 5633
(1999).
34. L. Mu, A. Chrastina, T. Levchenko, and V. P. Torchilin, J. Biomed.
Nanotechnol. 1, 190 (2005).
35. Y. L. Wang, B. K. Brittany, and I. Joseph, J. Biomed. Nanotechnol.
8, 751 (2012).
36. R. Surolia, P. Ranu, G. Manendra, and P. C. Ghosh, J. Biomed.
Nanotechnol. 8, 172 (2012).
. L. Li, Y. Zhang, S. Han, Z. Qu, J. Zhao, Y. Chen, Z. Chen, J. Duan,
Y. Pan, and X. Tang, J. Biomed. Nanotechnol. 7, 704 (2011).
. S. Nimesh, N. Gupta, and R. Chandra, J. Biomed. Nanotechnol.
7, 504 (2011).
. Y. Deng, L. Xu, X. Zeng, Z. Li, B. Qin, and N. He, J. Biomed.
Nanotechnol. 6, 318 (2010).
. Y. Deng, N. He, L. Xu, X. Li, S. Li, Z. Li, and H. Liu, Adv. Sci.
Lett. 4, 442 (2011).
. B. Koppolu, Z. Bhavsar, A. S. Wadajkar, S. Nattama, M. Rahimi,
F. Nwariaku, and K. T. Nguyen, J. Biomed. Nanotechnol. 8, 983
(
2012).
1
0. J. Lalani, Y. Raichandani, R. Mathur, M. Lalan, K. Chutani, A. K.
37. X. Y. Xiong, K. C. Tam, and L. H. Gan, J. Control Release 108, 263
(2005).
Mishra, and A. Misra, J. Biomed. Nanotechnol. 8, 918 (2012).
Received: 30 December 2012. Accepted: 4 July 2013.
4
850
J. Nanosci. Nanotechnol. 14, 4843–4850, 2014