# Synthesis of PET-PLA/Drug Nanoparticles and Their  Effect with Gold Nanoparticles for Controlled Drug Release  in Cancer Chemotherapy

Authors: K. Sathish Kumar; V. Selvaraj; M. Alagar
Journal: Research Letters in Nanotechnology
 (2008)

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## Abstract

Polyethylene terephthalate-polylactic acid copolymer (PET-PLA) was synthesized from bis (2-hydroxyethyl terephthalate) and L-lactic acid oligomer in the presence of manganese antimony glycoxide as a catalyst. The synthesized PET-PLA copolymer was used for controlled drug release systems with gold nanoparticles. Fluorouracil containing PET-PLA nanocapsules was prepared in the presence of gold nanoparticles by solvent evaporation method. The morphologies of the nanocapsules were characterized using scanning electron microscopy and transmission electron microscopy. Controlled release of Fu and Fu@Au was carried out in 0.1 M phosphate buffer (pH 7.4) and 0.1 M HCl solution. The results indicated that the drug release for gold nanoparticles/fluorouracil (Au@Fu) incorporated PET-PLA nanocapsules was controlled and slow compared to Fu incorporated PET-PLA nanocapsules. This may be due to the interaction between the gold nanoparticles and fluorouracil in PET-PLA nanocapsules.


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## Body

## 1. Introduction

Nanotechnology provides a
novel route for many biomedical applications especially in the case of
incurable diseases such as cancer, diabetes, and so forth. Cancer can affect
just every organ in human body. The various treatments of cancer are
chemotherapy, radiation therapy, surgery, biological therapy, hormone, and gene
therapies. Chemotherapy uses chemical agents (anticancer or cytotoxic drugs) to
interact with cancer cells to eradicate or control the growth of cancer.
Depending on the type of cancer and kind of drug used, chemotherapy drugs may
be administered differently. 5-Fluorouracil (5-Fu) is one of the oldest
chemotherapy drugs and has been used for decades. It is an active medicine
against many cancers. Over the past 20 years, increased understanding of the
mechanism of action of 5-Fu has led to the development of strategies that
increase its anticancer activity. 5-Fu is given for treatment of cancers like
bowel, breast, stomach, and gullet cancer [1]. However, anticancer drugs
normally attack both normal cells and cancerous cells when the drug was given
as an injection or tablet form for a long time. In order to over come this side
effect, targeting the drug delivery and sustained release of drugs are
required. Many research investigations are focused in the preparation of drug
encapsulated polymer nanoparticles for the controlled release applications.
Biodegradable polymers have become increasingly important in the development of
drug delivery systems [2–5]. There are several methods that can be used to make
microcapsules [6] from biodegradable polymers. Polyethylene terephthalate (PET)
is a semicrystalline polymer with high mechanical strength and an excellent
thermal stability. Copolyesters such as PET-PLA are biodegradable materials used
for tissue engineering, bone reconstruction, and controlled drug delivery
systems [7–9]. Further gold nanoparticles play an important role in cancer
therapy to detect or to deliver the drug to the cancerous cell without
affecting the normal cells and have good ability to form complex with many
drugs through chemical bonding. Nanoparticles have uniform shape and size and are
soluble in an aqueous medium. With this view in mind, the present work is
undertaken to synthesize PET-PLA copolyester and the preparation of PET-PLA/Fu
and PET-PLA/Fu-Au nanocapsules to study their drug release behavior. A
comparative study of sustained release of 5-Fu under different
pH conditions was
also carried out. The present work is the continuation of our previous work
with gold nanoparticles [10, 11].


## 2. Experimental

### 2.1. Synthesis of PET-PLA Copolyester

Bis
(2-hydroxy ethyl terephthalate) and L-lactic acid
oligomers (30/70) wt% were reacted in the presence of 10 mg of manganese
antimony glycoxide catalyst [12]. The reaction mixture was placed [13] in a 500 mL flask connected to a vacuum line (0.4 kPa) and immersed in an oil bath at
180°C for 6 hours. As the reactants were stirred, glycol and water were slowly
distilled out. The copolyesters were dissolved in chloroform, precipitated in
methanol, filtered and dried at 70°C. This was used without further
purification.


### 2.2. Preparation of Citrate-Capped Gold Nanoparticles

Trisodium
citrate (38.8 mM, 50 cm3) was added to a boiling HAuCl4 solution
(1 mM, 500 cm3). As a result, the previously yellow solution of gold
chloride turned to wine red color and gave a characteristic absorbance at 518 nm in the UV-vis spectrum.


### 2.3. Preparation of Nanocapsules

Nanocapsules were prepared by the solvent evaporation method
similar to that reported previously by Beck et al. [14]. For PET/PLA/5Fu-Au
experiment, 5-Fluorouracil (50 mg) was added in 0.5 mL nanogold aqueous
suspension (with a 30-minute shaking under ultrasound to help the adsorption of
5-Fluorouracil on gold nanoparticles) and this solution was added to an organic
polymer solution (300 mg PET-PLA + 5 mL CH2Cl2)
under stirring condition. This was continued until the organic solvent
was completely evaporated. The suspension became clear after all the
nanocapsules precipitated out of the solution. These nanocapsules were
collected by filtration and washed with deionized water to remove any
undesirable residuals. Finally, the clean nanocapsules were dried in a vacuum
oven at 40°C for 24 hours to ensure a complete removal of the organic solvent
and deionized water. All the nanocapsules were stored in a desiccator at 25°C. PET-PLA/Fu nanocapsules were also prepared
under similar conditions.Encapsulation efficiency
(%) = [(Mass of drug added during Nanoparticle (NP) preparation− Mass of free
drug in supernatant)/Mass of Drug added during NP preparation] × 100.Drug loading (%) = [Mass
of 5-Fluouracil in NP/Mass of NP recovered]× 100.


## 2.1. Synthesis of PET-PLA Copolyester

Bis
(2-hydroxy ethyl terephthalate) and L-lactic acid
oligomers (30/70) wt% were reacted in the presence of 10 mg of manganese
antimony glycoxide catalyst [12]. The reaction mixture was placed [13] in a 500 mL flask connected to a vacuum line (0.4 kPa) and immersed in an oil bath at
180°C for 6 hours. As the reactants were stirred, glycol and water were slowly
distilled out. The copolyesters were dissolved in chloroform, precipitated in
methanol, filtered and dried at 70°C. This was used without further
purification.


## 2.2. Preparation of Citrate-Capped Gold Nanoparticles

Trisodium
citrate (38.8 mM, 50 cm3) was added to a boiling HAuCl4 solution
(1 mM, 500 cm3). As a result, the previously yellow solution of gold
chloride turned to wine red color and gave a characteristic absorbance at 518 nm in the UV-vis spectrum.


## 2.3. Preparation of Nanocapsules

Nanocapsules were prepared by the solvent evaporation method
similar to that reported previously by Beck et al. [14]. For PET/PLA/5Fu-Au
experiment, 5-Fluorouracil (50 mg) was added in 0.5 mL nanogold aqueous
suspension (with a 30-minute shaking under ultrasound to help the adsorption of
5-Fluorouracil on gold nanoparticles) and this solution was added to an organic
polymer solution (300 mg PET-PLA + 5 mL CH2Cl2)
under stirring condition. This was continued until the organic solvent
was completely evaporated. The suspension became clear after all the
nanocapsules precipitated out of the solution. These nanocapsules were
collected by filtration and washed with deionized water to remove any
undesirable residuals. Finally, the clean nanocapsules were dried in a vacuum
oven at 40°C for 24 hours to ensure a complete removal of the organic solvent
and deionized water. All the nanocapsules were stored in a desiccator at 25°C. PET-PLA/Fu nanocapsules were also prepared
under similar conditions.Encapsulation efficiency
(%) = [(Mass of drug added during Nanoparticle (NP) preparation− Mass of free
drug in supernatant)/Mass of Drug added during NP preparation] × 100.Drug loading (%) = [Mass
of 5-Fluouracil in NP/Mass of NP recovered]× 100.


## 3. Results and Discussion

### 3.1. UV-vis Characterization

Figure1 shows the UV-visible
spectrum of citrate stabilized gold nanoparticles. The plasmon band observed for the wine red colloidal gold at 518 nm
is characteristic of the gold nanoparticles. The pure drug shows a maximum at
273 nm, but with the addition of 5-Fu to colloidal gold, both the bands at 273
and 518 nm pertaining to pure drug and Au colloids decrease in intensity
steadily with time.(a) and (b) show the UV-spectrum of Au nanoparticles and Fu@Au nanoparticles.
(a)(b)This decrease
is accompanied by the emergence of an additional peak at 650 nm (Figure1(b)), that is, a change from wine red to
blue with the addition of drug to colloidal gold. The appearance of the new
peak is due to the aggregation of gold nanoparticles and the replacement of
citrate by 5-Fu, leading to the formation of gold-drug complex. Citrate ions
are readily replaced by the –NH ligand on the surface of gold nanoparticles.
This ligand exchange reaction provides an important means for the chemical
functionalization of the nanoparticles and greatly extends the versatility of
these systems.


### 3.2. Morphological Characterization

PET-PLA/5-Fu-Au
and PET-PLA/5-Fu nanocapsules were easily distinguished from PET-PLA/5-Fu
nanocapsules by their color. While the color of PET-PLA/5-Fu nanocapsules was
white, PET-PLA/5-Fu-Au nanocapsules was purple/blue in color, due to the presence
of the gold nanoparticles.From the TEM (Figures2(a) and 2(b))
measurements, the average diameters of the gold nanoparticles were found to be
in the range of 18–20 nm.The TEM images of (a) Au@Fu, and (b) Au-Fu/PET-PLA nanoparticles.
(a)(b)Morphology of
nanocapsules (PET-PLA/5-Fu and PET-PLA/5-Fu-Au) prepared
was characterized by the SEM analysis (Figures3(a) and 3(b)).The SEM images of (a) Fu/PLA-PET
nanoparticles, and (b) Fu-Au/PET-PLA nanoparticles.
(a)(b)From the SEM measurements,
the size of nanocapsules was found to
be in the range of 230–260 nm. The average sizes of the microcapsules
to nanocapsules can be prepared by changing the rotational speed from 2000 to
12000 rpm.The
average size decreased with an increase in the rotational speed, because of the
higher shear stress. However, since the size difference was within the limits
of statistical error, this statement remained speculative without further
investigations. The surface topography of PET-PLA/5-Fu and PET-PLA/5-Fu-Au
nanocapsules was smooth as seen in SEM photographs. The SEM micrographs
manifested that nanocapsules prepared in the present study possess a nearly
spherical shape.


### 3.3. Encapsulation Efficiency

The
encapsulations of PET-PLA/5-Fu-Au and PET-PLA/5-Fu nanocapsules were obtained
both in the presence and in the absence of gold nanoparticles. For PET-PLA/5-Fu-Au nanocapsules, the adsorption
of 5-Fluorouracil on Au led to a larger particle (Fu-Au complex) size and made
the diffusion of 5-Fluorouracil to the external solution less efficient. Gold
nanoparticles also hindered the diffusion of 5-Fluorouracil to the external
solution because of their insolubility in water and methylene chloride. In
addition, each gold nanoparticle was composed of many Au atoms and would adsorb
more than one 5-Fluorouracil molecules. As a result, PET-PLA/5-Fu-Au
nanocapsules had a higher encapsulation efficiency (70.21%) and a larger
average size than PET-PLA/5-Fu nanocapsules (42.56%). The amount of 5-Fluorouracil
entrapped within NP was determined by measuring the amount of nonentrapped drug
in the supernatant recovered after centrifugation and washing of the
nanoparticles, using a UV-visible spectrophotometer [15].However, PET-PLA/5-Fu-Au
nanocapsules had a higher drug loading
(4.63%) than PET-PLA/5-Fu nanocapsules (2.46%)


### 3.4. Drug Release Study

The release profiles of
5-Fluorouracil from PET-PLA/5-Fu and PET-PLA/5-Fu-Au nanocapsules in the
hydrochloric acid (0.1M) and the phosphate buffered saline (pH 7.4) at 37 ± 0.1°C are shown in Figure 4. Desorption profiles
were obtained as follows. 0.03 g of drug encapsulated polymer nanoparticles was
mixed with 10 mL of phosphate buffer solution (pH 7.4) in five fractions. Each
fraction was centrifuged as a function of time. The absorbance of each solution
was monitored at different times. One sample solution was used only once to
ensure that there was no change in the concentration of the solution.Figure 4
The controlled drug release of Fu@Au in 0.1 M HCl, and 0.1 M PBS.The
intensity of absorption was plotted against time which gave the desorption
profile of fluorouracil. In the present study, fluorouracil encapsulated
PET-PLA in the presence of gold nanoparticles was used for controlled drug release.
In order to find the drug loading, a standard graph was drawn using known
concentration of the drug. Figure4 shows the drug release at different
intervals of time using phosphate buffer solution at pH 7.4. The same procedure
has been repeated for the drug encapsulated with gold nanoparticles. From
Figure 4, it was understood that the drug release was slow and sustained. The
release rate of 5-Fluorouracil for PET-PLA/5-Fu-Au nanocapsules was slower than
that of PET-PLA/5-Fu nanocapsules in the two different dissolution media.
PET-PLA/5-Fu-Au nanocapsules had a slower release behavior mainly because gold
nanoparticle in nanocapsules hindered the diffusion of 5-Fluorouracil away from
the nanocapsules. An initial burst effect (i.e., the rapid release of 5-Fluorouracil)
of PET-PLA/5-Fu nanocapsules was observed as shown in Figure 4.The
effect of gold nanoparticles on the release rate was particularly important because
the release rates of 5-Fluorouracil for PET-PLA/5-Fu nanocapsules were higher
than that of PET-PLA/5-Fu-Au nanocapsules in two different dissolution media.
Further, the release rates of 5-Fluorouracil from PET-PLA/5-Fu and
PET-PLA/5-Fu-Au nanocapsules were relatively higher in 7.4 PBS (0.1 M) than in pH 0.1 M HCl. This observation was
attributed to the fact that the dissolution medium had a strong influence on the solubility of the drug, and
the solubility of 5-Fluorouracil in pH 7.4 PBS was higher than that in 0.1 M HCl
at the same temperature.


## 3.1. UV-vis Characterization

Figure1 shows the UV-visible
spectrum of citrate stabilized gold nanoparticles. The plasmon band observed for the wine red colloidal gold at 518 nm
is characteristic of the gold nanoparticles. The pure drug shows a maximum at
273 nm, but with the addition of 5-Fu to colloidal gold, both the bands at 273
and 518 nm pertaining to pure drug and Au colloids decrease in intensity
steadily with time.(a) and (b) show the UV-spectrum of Au nanoparticles and Fu@Au nanoparticles.
(a)(b)This decrease
is accompanied by the emergence of an additional peak at 650 nm (Figure1(b)), that is, a change from wine red to
blue with the addition of drug to colloidal gold. The appearance of the new
peak is due to the aggregation of gold nanoparticles and the replacement of
citrate by 5-Fu, leading to the formation of gold-drug complex. Citrate ions
are readily replaced by the –NH ligand on the surface of gold nanoparticles.
This ligand exchange reaction provides an important means for the chemical
functionalization of the nanoparticles and greatly extends the versatility of
these systems.


## 3.2. Morphological Characterization

PET-PLA/5-Fu-Au
and PET-PLA/5-Fu nanocapsules were easily distinguished from PET-PLA/5-Fu
nanocapsules by their color. While the color of PET-PLA/5-Fu nanocapsules was
white, PET-PLA/5-Fu-Au nanocapsules was purple/blue in color, due to the presence
of the gold nanoparticles.From the TEM (Figures2(a) and 2(b))
measurements, the average diameters of the gold nanoparticles were found to be
in the range of 18–20 nm.The TEM images of (a) Au@Fu, and (b) Au-Fu/PET-PLA nanoparticles.
(a)(b)Morphology of
nanocapsules (PET-PLA/5-Fu and PET-PLA/5-Fu-Au) prepared
was characterized by the SEM analysis (Figures3(a) and 3(b)).The SEM images of (a) Fu/PLA-PET
nanoparticles, and (b) Fu-Au/PET-PLA nanoparticles.
(a)(b)From the SEM measurements,
the size of nanocapsules was found to
be in the range of 230–260 nm. The average sizes of the microcapsules
to nanocapsules can be prepared by changing the rotational speed from 2000 to
12000 rpm.The
average size decreased with an increase in the rotational speed, because of the
higher shear stress. However, since the size difference was within the limits
of statistical error, this statement remained speculative without further
investigations. The surface topography of PET-PLA/5-Fu and PET-PLA/5-Fu-Au
nanocapsules was smooth as seen in SEM photographs. The SEM micrographs
manifested that nanocapsules prepared in the present study possess a nearly
spherical shape.


## 3.3. Encapsulation Efficiency

The
encapsulations of PET-PLA/5-Fu-Au and PET-PLA/5-Fu nanocapsules were obtained
both in the presence and in the absence of gold nanoparticles. For PET-PLA/5-Fu-Au nanocapsules, the adsorption
of 5-Fluorouracil on Au led to a larger particle (Fu-Au complex) size and made
the diffusion of 5-Fluorouracil to the external solution less efficient. Gold
nanoparticles also hindered the diffusion of 5-Fluorouracil to the external
solution because of their insolubility in water and methylene chloride. In
addition, each gold nanoparticle was composed of many Au atoms and would adsorb
more than one 5-Fluorouracil molecules. As a result, PET-PLA/5-Fu-Au
nanocapsules had a higher encapsulation efficiency (70.21%) and a larger
average size than PET-PLA/5-Fu nanocapsules (42.56%). The amount of 5-Fluorouracil
entrapped within NP was determined by measuring the amount of nonentrapped drug
in the supernatant recovered after centrifugation and washing of the
nanoparticles, using a UV-visible spectrophotometer [15].However, PET-PLA/5-Fu-Au
nanocapsules had a higher drug loading
(4.63%) than PET-PLA/5-Fu nanocapsules (2.46%)


## 3.4. Drug Release Study

The release profiles of
5-Fluorouracil from PET-PLA/5-Fu and PET-PLA/5-Fu-Au nanocapsules in the
hydrochloric acid (0.1M) and the phosphate buffered saline (pH 7.4) at 37 ± 0.1°C are shown in Figure 4. Desorption profiles
were obtained as follows. 0.03 g of drug encapsulated polymer nanoparticles was
mixed with 10 mL of phosphate buffer solution (pH 7.4) in five fractions. Each
fraction was centrifuged as a function of time. The absorbance of each solution
was monitored at different times. One sample solution was used only once to
ensure that there was no change in the concentration of the solution.Figure 4
The controlled drug release of Fu@Au in 0.1 M HCl, and 0.1 M PBS.The
intensity of absorption was plotted against time which gave the desorption
profile of fluorouracil. In the present study, fluorouracil encapsulated
PET-PLA in the presence of gold nanoparticles was used for controlled drug release.
In order to find the drug loading, a standard graph was drawn using known
concentration of the drug. Figure4 shows the drug release at different
intervals of time using phosphate buffer solution at pH 7.4. The same procedure
has been repeated for the drug encapsulated with gold nanoparticles. From
Figure 4, it was understood that the drug release was slow and sustained. The
release rate of 5-Fluorouracil for PET-PLA/5-Fu-Au nanocapsules was slower than
that of PET-PLA/5-Fu nanocapsules in the two different dissolution media.
PET-PLA/5-Fu-Au nanocapsules had a slower release behavior mainly because gold
nanoparticle in nanocapsules hindered the diffusion of 5-Fluorouracil away from
the nanocapsules. An initial burst effect (i.e., the rapid release of 5-Fluorouracil)
of PET-PLA/5-Fu nanocapsules was observed as shown in Figure 4.The
effect of gold nanoparticles on the release rate was particularly important because
the release rates of 5-Fluorouracil for PET-PLA/5-Fu nanocapsules were higher
than that of PET-PLA/5-Fu-Au nanocapsules in two different dissolution media.
Further, the release rates of 5-Fluorouracil from PET-PLA/5-Fu and
PET-PLA/5-Fu-Au nanocapsules were relatively higher in 7.4 PBS (0.1 M) than in pH 0.1 M HCl. This observation was
attributed to the fact that the dissolution medium had a strong influence on the solubility of the drug, and
the solubility of 5-Fluorouracil in pH 7.4 PBS was higher than that in 0.1 M HCl
at the same temperature.


## 4. Conclusion

The binding of
fluorouracil to colloidal gold and encapsulation of Fu to polymer nanoparticles
were studied using different analytical techniques. The aggregations of gold
nanoparticles were ascertained using UV-visible spectroscopy and TEM analysis.
Further, it was observed that the drug release was slow and sustained in the
case of PLA-PET/Au-Fu compared to that of PLA-PET/Fu nanoparticles due to the interaction
between the drug and gold nanoparticles. The combination of gold and polymer
with Fu results in a more potent complex compared to the individual parts,
since such a complex can release the drug at controlled rate as well as at the
targeted places.



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*Source: 389512-2008-07-02.xml*