Koushik et al: Development of Solid-Self Micron Emulsifying Drug Delivery Systems
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International Journal of Pharmaceutical Sciences and Nanotechnology
Volume 6 • Issue 2 • July– September 2013
IJPSN-9-30-12-CHANDRETHIYA
Research Paper
Development and Characterization Colchicine-Loaded PEGylated
Gelatin Nanoparticles for Targeted Delivery to Tumor
G.D. Chandrethiya, P.K. Shelat* and M.N. Zaveri
K.B. Institute of Pharmaceutical Education and Research, GH-6, Sector 23, Gandhinagar-380023, Gujarat, India.
Received September 9, 2012; accepted February 15, 2013
ABSTRACT
PEGylated gelatin nanoparticles loaded with colchicine were
prepared by ethanol precipitation method. Poly-(ethylene
glycol)-5000-monomethylether (MPEG 5000), a hydrophilic
polymer, was used to pegylate gelatin. Gluteraldehyde was
used as cross-linking agent. To obtain a high quality product,
major formulation parameters were optimized.
Spherical
particles with mean particles of 193 nm were measured by a
Malvern particle size analyzer. Entrapment efficiency was
found to be 71.7 ± 1.4% and determined with reverse phase
high performance liquid charomatography (RP-HPLC). The in
vitro drug release study was performed by dialysis bag method
for a period of 168 hours. Lyophilizaton study showed sucrose
at lower concentrations proved the best cryoprotectant for this
formulation. Stability study revealed that lyophilized
nanoparticles were equally effective (p < 0.05) after one year of
storage at 2-8°C with ambient humidity. In vitro antitumoral
activity was accessed using the MCF-7 cell line by MTT assay.
The IC50 value was found to be 0.034 μg/ml for the prepared
formulation. The results indicate that PEGylated gelatin
nanoparticles could be utilized as a potential drug delivery for
targeted drug delivery of tumors.
KEYWORDS: Nanoparticles; PEGylation; anticancer drug; colchicine, gelatin.
Introduction
Nanoparticles are solid particles or particulate
dispersions with a size in the range of 10-1000 nm. The
drug is dissolved, entrapped, encapsulated, or attached
to a nanoparticle matrix (Gibaldi et al., 2006). In tumors,
targeted polymeric NPs can be used to deliver
chemotherapies to tumor cells with greater efficacy and
decreased cytotoxicity on peripheral healthy tissues
(Chan et al., 2010). Preventing harmful side effects,
minimizing drug degradation and loss, increasing drug
bioavailability and the fraction of the drug accumulated
in the required zone, and improving various drug
deliveries and drug targeting systems are goals currently
under development. A good nanoparticle formulation
should have a high drug-loading capacity, which reduces
the quantity of matrix materials for administration
(Bhadra et al., 2002; Lee et al., 2005). Drug loading can
be done by two methods: the incorporation method
(incorporating at the time of nanoparticles production)
and adsorption/absorption technique (absorbing the drug
after formation of nanoparticles by incubating the carrier
with a concentrated drug solution) (Desai et al., 1996;
Kroil et al., 1998; Kreuter et al., 2003).
The aim of present study was to prepare long
circulating gelatin nanoparticles and achieve the
targeted delivery to tumors. Tumor blood vessels are
generally characterized by poor lymphatic drainage and
leaky vasculature. Because of these properties,
nanoparticles are easily entrapped and not removed
efficiently. Thus, nanoparticles entrapped in the tumor
target using a passive targeting mechanism which is
called the EPR effect (Lisa et al., 2004). The modification
of a protein, peptide, or non-peptide molecule by the
linking of one or more polyethylene (PEG) chain(s) is
defined as PEGylation.
MPEG-5000 is an FDA
approved, non-toxic, non-antigenic, non-immunogenic,
and highly water soluble polymer. Such modifications
with PEG either with drug or polymer have several
advantages like prolong residence in the body, decreased
degradation by metabolic enzymes, and reduction or
elimination of protein immunogenicity (Gref et al., 1996).
PEGylation prevents unwanted segregation due to
secondary interaction between nanoparticles. Also,
PEGylation of nanoparticles minimizes recognition by
proteins and cells in the body, allowing long circulation
and increasing the possibility of reaching the target
(Donald et al., 2006). Colchicine is a recently detected
anticancer drug. Colchicine interacts with tubulin and
perturbs the assembly dynamics of microtubules. Though
its use has been limited because of its toxicity, colchicine
can still be used as a lead compound for the generation of
potent anti-cancer drugs. Colchicine binds to tubulin in a
poorly reversible manner with high activation energy
(Sarkar and Yang, 2010).
Materials and Methods
Material
Colchicine was gifted from Cadila Healthcare
Limited. Poly-(ethylene glycol)-5000 monomethylether
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Chandrethiya et al: Development and Characterization Colchicine Loaded PEGylated Gelatin Nanoparticles for Cancer Therapy 2059
(MPEG) was procured from Sigma- Aldrich Cheme,
Germany. Gelatin (bloom strength 225) was obtained
from Troikaa Pharmaceutical Limited, Ahmedabad. All
the reagents were of analytical grade.
PEGylation of Gelatin
1. To prepare intermediate PEG epoxide
MPEG was dissolved in dimethyl formamide.
Triethylamine and epichlorhydrine were added in
molar ratio of 2:1. The mixture was kept in an oil
bath for 18 hrs at 40°C. It was precipitated with
cold dry ethyl ether and filleted.
2. Grafting of gelatin with activated PEG
PEG-epoxide was dissolved in alkaline borate
buffer pH 8.5 and a known quantity of gelatin was
added in it at 40°C. It was grafted for 18 hours in
different proportions and precipitated with three
fold excess of acetone. The mixture was purified
with dialysis against deionized distilled water
with a membrane cut of 12k DA – 14k DA Hi
media for 24 hrs. The dispersant was filled in a
vial and lyophilized. The percentage of
PEGylation was optimized by taking different
proportions of PEG epoxide and polymer. The %
PEGylation was measured by a UV visible
spectrophotometer
(Shimadzu
UV-1700,
Shimadzu Corporation, Japan) at λmax 422nm.
Preparation of nanoparticles
Nanoparticles were prepared by the ethanol
precipitation method. PEGylated gelatin was dissolved in
deionized distilled water at 37-40°C and pH was adjusted
with sodium chloride up to 7.0. Dropwise additions of
ethanol to the gelatin solution were done to precipitate
PEGylated gelatin nanoparticles. Glutaraldehyde (25%)
was added dropwise to cross link nanoparticles. The
ethanol to water ratio was optimized (7:2).
Characterization of PEGylated Gelatin
Nanoparticles
Characterization of PEGylated Gelatin
An amino group was derivatized from a
trinitrophenylation reaction with dye (trinitrobenzene
sulphonic acid). Each solution was made 1 mg/ml and 10
ml of resulting solution was mixed with 0.25 ml of dye
solution. Absorbance was taken at 420 nm and %
derivatization was calculated.
Particle size measurement
Particle size measurement of colchicine-loaded
PEGylated gelatin nanoparticles were carried out by
dynamic light scattering using Malvern Hydro 2000 SM
particle size analyzer (Malvern Instruments UK).
Nanodispersion was diluted to measure the size of
particles and polydispersibility index. Nanodispersion
was injected through sample injection port and the laser
obscuration range was maintained between 2-20%.
Drug entrapment efficiency
The % drug entrapment efficiency of PEGylated
nanoparticles was found using an Ultracentrifuge (REMI
centrifuge). The nanoparticle suspension was subjected
to 20,000 rpm. The supernant was collected and analyzed
for the free drug for the quantitative determination of
colchicine using reverse phase high performance liquid
chromatography (RP HPLC) (LC 2010C HT, CLASS VP
Software).
% entrapment =
Total amount of drug added – Amount of free drug
×100
Total amount of drug addded
In vitro release study
In-vitro release rate of nanoparticles was evaluated
by the dialysis bag method in pH 7.4 phosphate buffer
saline, up to the 168 hr incubation period. The
nanoparticle suspension equivalent to 2 mg of colchicine
was placed in a cellulose dialysis bag (Cut off 12000 Hi
Media) and sealed at both ends. The dialysis bag, which
acts as a donor compartment, was immersed in the
receptor compartment containing 200 ml of diffusion
medium which was stirred at medium speed and
maintained at 37±2ºC. The receptor compartment was
covered to prevent the evaporation of diffusion medium.
Samples were withdrawn at regular time intervals and
the same volume was replaced by fresh diffusion
medium. The samples were analyzed (simultaneous
analysis) using RP HPLC.
Lyophilization
The colchicine-loaded PEGylated nanoparticles were
subjected to lyophilization study. The prepared
nanoparticles were purified by an Ultracentrifuge.
Sucrose, mannitol, and dextrose were selected as
cryoprotectants. The nanoparticle: cryoprotectant ratio
was selected in 1:1, 1:2, 1:3 proportions for their
cryoprotectant efficiency. Here, the cryoprotectant was
dissolved in water. The cryoprotectant solution and
nanoparticle suspension was mixed in 2 ml and 8 ml,
respectively. Each set of mixture was frozen in a deep
freezer at -20°C. The samples were vacuum-dried in
Vertix lab equipment (GENESIS 25EL85) below 27°C for
24 hrs. Lyophilized nanoparticles were evaluated by
particle size analysis, flowability, and redispersibilty.
Stability Study
Prepared nanoparticles were subjected to stability
studies in triplicate at conditions of 2-8°C with ambient
humidity and 30+2°C/60+5% humidity condition for the
period of six months.
Cytotoxicity Study
MCF-7 (Breast Cancer cell line), was procured from
National center for cell sciences, DBT, Pune, India. MTT
Assay 3-(4,5-dimethyl thiazole-2yl)-2, diphenyl tetrazolium) was performed. Colchicine concentrations were 2,
1, 0.2, 0.02, and 0.002 µg/ml. MCF-7 cells were grown in
DMEM
(Dulbecco’s
Modified
Eagle
Medium)
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Int J Pharm Sci Nanotech
supplements with 10% (v/v) fetal bovine serum and
100IU/ml of Penicillin G sodium and 100 µg/ml
Streptomycin sodium. Cells were incubated with 5%
O2/95% air humidified atmosphere at 37°C. Cells were
seeded in a 96 well plate at a density of 2500 viable cells.
They were further incubated for 24 h to allow cells to
attach. Cells incubated with plain nanoparticles and
drug-loaded nanoparticles for 0, 1, 2, and 3 days. At the
determined time, the formulation was replaced with
DMEM containing MTT (5 mg/ml) and cells were
incubated for additional 4 hrs. MTT was aspirated off
and dimethyl sulphoxide (DMSO) was added to dissolve
formazan crystals. Using a BioRad microplate reader,
absorbance was measured at 570 nm. Untreated cells
were used as the control with 100% viability. Cells
without the MTT addition were taken as blank to
calibrate the spectrophotometer to zero absorbance. For
positive control for cytotoxicity, Triton X-100 1% was
used. Final results were calculated as mean values ± SD
of five measurements.
Results
PEGylation of Gelatin
In order to optimize preparation of colchicine-loaded
PEGylated gelatin nanoparticles, the ratio of ethanol and
water was optimized. PEGylation was performed in two
steps and PEG-epoxide was made and was confirmed by
thin layer chromatography. Thin layer chromatography
was performed using chloroform: methanol as the mobile
phase. A distinct spot was observed and 94.6% yield was
obtained. Activated PEG was grafted with free amino
group of gelatin. The PEG-epoxide and gelatin ratio was
optimized to achieve maximum PEGylation. In our study,
65.77% PEGylation was achieved. The optimum amount
of % PEGylation can be achieved by changing the MPEGEpoxide to gelatin ratio as shown in Table 1.
TABLE 1
Different proportion of PEG- epoxide:
gelatin to optimize % PEGylation.
PEG-epoxide: gelatin
% PEGylation
0.5:1
28.36%
1:1
43.38%
Vol 6; Issue 2 • July−September 2013
In vitro drug release study
The % drug release of colchicine-loaded gelatin
nanoparticles and colchicine-loaded PEGylated gelatin
nanoparticles were 98.23% and 80.03% respectively after
168 hrs.
Lyophilization study and stability study
Sucrose with nanoparticles in a 1:1 proportion was
better than all other cryoprotectants. After 6 months of
stability study, results showed that nanoparticles are
stable at 2-8°C with ambient humidity.
Discussion
Colchicine-loaded PEGylated nanoparticles were
prepared from a naturally-obtained hydrophilic,
biodegradable, and easily available polymer, gelatin. The
method of preparation of gelatin nanoparticles includes a
lower concentration of gluteraldehyde. Therefore, the
overall formulation does not include risk of toxic
materials. Hence, such formulations are robust. The size
of nanoparticles is helpful to target tumor tissue with the
EPR effect. The most peripheral human tumor has an
effective pore size range from 200 nm to 600 nm, with a
mean size of about 400 nm. Based on the cutoff size of
tumors, the EPR effect allows passive targeting to
tumors (Yuan et al., 1995). It can be justified that the
formulations have better targeting potentials. Hence, the
anticancer drug-loaded formulations have a reduction in
side effects of anticancer drugs. Optimized formulations
have better drug entrapment. The % entrapment of drug
is depends on amount of polymer used. As the drug:
polymer ratio increased above 1:12.5, the entrapment
efficiency lowered as shown in Table 2. More than 80% of
drug release from colchicine-loaded gelatin nanoparticles
and PEGylated colchicine-loaded gelatin nanoparticles
was shown by 168 hrs, while PEGylated colchicineloaded gelatin nanoparticles showed decrease in drug
release indicative of long circulating action of PEGylated
nanoparticles as shown in Fig. 2. MPEG or non-ionic
surfactants create hydrophilic coating, which improves
circulation time of nanoparticles (Gref et al., 1996).
Colchicine-loaded gelatin nanoparticles and colchicineloaded PEGylated gelatin nanoparticles displayed burst
release during 1 h (> 10%).
1.5:1
51.86%
2:1
65.77%
TABLE 2
2.5:1
53.50%
3:1
33.44%
Different proportion of drug: polymer ratio to optimize %
drug entrapment efficiency.
Particle Size and % drug entrapment
The particle size of colchicine-loaded gelatin
nanoparticles was 190 nm and that of PEGylated
nanoparticles was 193nm. Scanning Electron Microscopy
(SEM) (JSM, 5610 LV, JEOL, Datum, Ltd., Japan) study
showed spherical shaped particles. The drug entrapment
efficiency was found to be 71.7 ± 1.4%.
Drug: polymer ratio (%w/w)
% Entrapment
1:5
1:7.5
1:10
1:12.5
1:15
35.1± 0.9%
67.1± 1.8%
71.7 ± 1.4%
66.3± 2.1%
54.7± 1.3%
Chandrethiya et al: Development and Characterization Colchicine Loaded PEGylated Gelatin Nanoparticles for Cancer Therapy 2061
Fig. 1. PEGylation of gelatin
nanoparticles.
Fig. 2. Percent drug release of
colchicine-loaded PEGylated gelatin
nanoparticles and colchicine-loaded
gelatin nanoparticles. ♦ % Drug release of Col-Gel-NPs
■% Drug release of Col-PEG-Gel-NPs
Col-GEL-NPS= Colchicine-loaded gelatin nanoparticles
Col-PEG-GEL-NPS= Colchicine-loaded gelatin nanoparticles
The burst phase was followed by hydration and
swelling of nano-matrix which eventually led to
controlled release profile lasting up to 168 hrs.
Hydration leads to an increase in the path length of
molecules and consequently the rate of their diffusion
becomes lower (Wong et al., 1999). The release profile of
colchicine-loaded PEGylated gelatin formulations showed
slow and gradual release compared to colchicine-loaded
non-PEGylated gelatin formulations. Linear curves were
not obtained upon plotting the % cumulative drug release
versus time for all drug-loaded PEGylated and nonPEGylated formulations, indicating that the release is
zero order. However, the regression coefficient of the plot
of Mt/M∞ versus the square root of time (t1/2) was found
to lie between 0.81 and 0.971, indicating that a linear
relationship exists between these two parameters and
that the release obeys Higuchi’s diffusion-controlled
model and also indicated the drug entrapped within
matrix (Seda et al., 2012) as shown in Fig. 3.
Usually, nanoparticles dispersion showed an increase
in particle size in a shorter period of time during storage.
Lyophilization provided physical and chemical stability
by preventing Ostwald ripening and hydrolysis reaction
(Mehnert and Mader, 2001). Cryoprotectants have been
used to decrease the aggregation of nanoparticles due to
stress during process of freeze drying (Shahgaldian et al.,
2003). Various cryoprotectants were screened at the
cryoprotectant to nanoparticles ratios of 1:1, 1:2, and 1:3.
All the cryoprotectants were evaluated based on change
in particle size compared to initial particle size as shown
in Table 3.
Sucrose with nanoparticles in a 1:1
proportion was better than all other cryoprotectants.
Furthermore, based on our results, flowability, and
redispersibility, sucrose was chosen as the best
cryoprotectant among all selected cryoprotectants for
further experiments as shown in Table 4. The physical
stability of nanoparticles was checked for 6 months at 28°C with ambient humidity and 30+2°C/60+5% humidity.
After 6 months of stability study, there was no
significant change in particle size at 2-8°C with ambient
humidity. There was significant change in particle size at
30+2°C/60+5% humidity as mentioned in Table 4. In
lyophilisation study, the cryoprotectant ratio was
optimized and the nanoparticle to sucrose ratio of 1:1
was found to be the best cryoprotectant among all ratios
of different cryoprotectants. Stability study concludes
drug-loaded PEGylated gelatin nanoparticles were stable
for 12 months at 2-8°C with ambient humidity. The
drug-loaded PEGylated gelatin nanoparticles obeyed zero
order kinetics and followed Higuchi’s diffusion controlled
model, also the drug entrapped within matrix.
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Int J Pharm Sci Nanotech
Vol 6; Issue 2 • July−September 2013
TABLE 4
Flow property and redispersibility of reconstituted PEGylated
gelatin nanoparticles after lyophilization
Cryoprotectant
Mannitol
Dextrose
Sucrose
Fig. 3. Mt/M∞ (PEG-ANA-GEL- NPS) →t1/2.
TABLE 3
Particle size analysis of reconstituted PEGylated gelatin
nanoparticles after lyophilization
Cryoprotectant
Mannitol
Dextrose
Sucrose
Ratio of (Nanoparticles:
cryoprotectant)
Particle size (nm)
1:1
1:2
1:3
1:1
1:2
1:3
1:1
1:2
1:3
335
351
374
367
326
316
195
211
248
-- Very poor
+ Good,
Ratio
1:1
1:2
1:3
1:1
1:2
1:3
1:1
1:2
1:3
PEGylated Gelatin Nanoparticles
Flowability
Redispersibiliy
+
-+
++
+
+
- Poor,
++ Very Good
--+
--++
+
+/+/- Moderately Good,
TABLE 5
Particle size determination PEGylated gelatin nanoparticles for a
period of 12 months at 2-8°C with ambient humidity and
30+2°C/60+5% humidity.
Average particle size (nm)
1
2
3
months
months
months
Batch
0
days
2-8°C with
ambient
humidity
30+2° C/60 +
5% humidity
195
(5.3)
198
(6.8)
203
(6.7)
214
(4.8)
224
(7.9)
195
(5.3)
227
(5.5)
268
(3.4)
334
(5.1)
376
(3.3)
6
months
Fig. 4.
Percent cell viability
increases with cconcentration
(µg/ml).
Conclusions
In vitro antitumor activity of blank nanoparticles,
drug-loaded nanoparticles and PEGylated drug-loaded
nanoparticles were evaluated by MTT assay using MCF7 cell line. Cell viability achieved 12.0% after 24 hrs at
2mg/ml concentration of drug, for colchicine-loaded
PEGylated gelatin nanoparticles, while lower colchicine
concentration achieved 62.3% cell viability after 24 hrs.
The IC50 value was found to be 0.034 μg/ml for 24 hrs.
Drug viability was totally suppressed after 48 hours of
incubation period at different concentrations with all
formulations. As the concentration lowered, more
incubation time was required to suppress the cell
viability. No cytotoxic effects were observed for drug-free
gelatin nanoparticles. During the incubation period, the
cytotoxic effect may be due to the free drug as well as
drug released from the nanoparticles (Fabienne et al.,
2009). Cytotoxicity study showed effects due to free drug
and
drug
entrapment
within
the
PEGylated
nanoparticles. Hence, our study proved the targeting
potential of polymeric nanoparticles loaded with
anticancer drug.
Chandrethiya et al: Development and Characterization Colchicine Loaded PEGylated Gelatin Nanoparticles for Cancer Therapy 2063
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Address correspondence to: G.D. Chandrethiya, K.B. Institute of
Pharmaceutical Education and Research, GH-6, Sector 23, Gandhinagar380023, Gujarat-India. Tel: +91 79 23249069, +91 79 232345270
Email: [email protected]