PLLA FIBERS SOAKED WITH GENTAMICIN SULFATE AS A DRUG DELIVERY SYSTEM

Abstract

Elektrospun poly(L-lactide) (PLLA) fibers with high surface area to volume ratio were
investigated to evaluate their potentional applications for biomedical devices and drug
delivery carriers. This study is focused on gentamicin sulfate (GS) release profiles for 240
hours time period in an environment simulating body fluids (phosphate-buffered solution,
PBS). To lead the gentamicin sulfate, electrospun fibers samples were simply individually
immersed in 3 mL of drug solution with concencration of 50 mg/ml for 12 hours. After that,
soaked electrospun fibres were removed from drug solution and dried under vacum for 24
hours. The samples were transfered to fresh PBS and incubated at 37 0C for 1, 2, 4, 8, 10, 24,
48, 72, 120, 168, 240 hours. Difference in weight of the sample before and after drug
saturation is the ratio of weight of absorbed drug per unit mass of soaked material.The amount
of gentamicin sulfate released from the samples was determinate using UV-VIS spectroscope.
In order to check whether the entire drug was released into the simulated body fluids scanning
electron microscopy observation of the surface of the electrospun fibers and FTIR
measurement were performed. The results indicated that almost 80 % of the drug absorbed on
the surface of the PLLA fibers was released during first 10 h of the experiment.

Full text

PLLA FIBERS SOAKED WITH GENTAMICIN SULFATE AS A DRUG
DELIVERY SYSTEM
A. WITECKA*, E. CHOIŃSKA*, E. KIJEŃSKA*, W. TOMASZEWSKI**,
W. ŚWIĘSZKOWSKI*, K. J. KURZYDŁOWSKI*
* Warsaw University of Technology, Faculty of Materials Science and Engineering, 141
Woloska Street, 02 507 Warsaw, Poland
** Institute of Biopolymers and Chemical Fibers, 19/27 Maria Skłodowska-Curie Street,
Łódź Poland
E-mail: ag.witecka@gmail.com
ABSTRACT
Elektrospun poly(L-lactide) (PLLA) fibers with high surface area to volume ratio were
investigated to evaluate their potentional applications for biomedical devices and drug
delivery carriers. This study is focused on gentamicin sulfate (GS) release profiles for 240
hours time period in an environment simulating body fluids (phosphate-buffered solution,
PBS). To lead the gentamicin sulfate, electrospun fibers samples were simply individually
immersed in 3 mL of drug solution with concencration of 50 mg/ml for 12 hours. After that,
soaked electrospun fibres were removed from drug solution and dried under vacum for 24
hours. The samples were transfered to fresh PBS and incubated at 37 0C for 1, 2, 4, 8, 10, 24,
48, 72, 120, 168, 240 hours. Difference in weight of the sample before and after drug
saturation is the ratio of weight of absorbed drug per unit mass of soaked material.The amount
of gentamicin sulfate released from the samples was determinate using UV-VIS spectroscope.
In order to check whether the entire drug was released into the simulated body fluids scanning
electron microscopy observation of the surface of the electrospun fibers and FTIR
measurement were performed. The results indicated that almost 80 % of the drug absorbed on
the surface of the PLLA fibers was released during first 10 h of the experiment.
Keywords: PLLA Nanofibers, Gentamicin Sulfate, DDS
1. INTRODUCTION
The replacement of defects in bone tissue caused by injuries, resections or infections is one of
the serious problems for tissue engineering today [1]. Polymeric fibers fabricated using
electrospinning method has a great potential in designing the tissue engineered scaffolds
because of their high surface area to volume ratio [2]. Electrospinning is a versatile technique
which uses electric field to fabricate fibers from polymeric solutions [3].
Nowadays, electrospun fibers are intensively investigated for applications such as biomedical
devices, tissue engineered scaffolds, and drug delivery systems (DDS), which are developed
to relaese drugs locally in the sites of implementation [2].
The aim of the study was to examine drug delivery system consist of poly(L-lactide) (PLLA)
fibrous mate soaked with gentamicin sulfate (GS). PLLA is well known as aliphatic polyester
and it is used in tissue engineering because of its appropriate biocompability, degradability
and mechanical properties [4] and gentamicin sulfate is an antibiotic used to treat many types
of infections, and often it is administered during orthopedic surgery [5].
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2. MATERIALS AND METHODS
2.1. Materials
The polymer PLLA was purchased from Boehringer-Ingelheim (Germany). Chloroform used
as a solvent was purchased from Chempur (Poland). Dimethyl sulfoxide (DMSO), gentamicin
sulfate and phosphate-buffered solution (PBS) were purchased from Sigma-Aldrich (USA).
2.2. Methods
2.2.1. Fibrous mats fabrication: PLLA was dissolved in Chloroform-DMSO solution (with
ratio of 85:15) and stirred for 24 h to obtain clear solution with concentration of 7%.
Electrospinning was performed using setup with 12 needles (each with diameter of 0.8 mm)
head for 6 h, with attached voltage of 12 kV.
2.2.2. Sample preparation: Polymeric fibrous mat was cut into pieces of equal size 10 x 10
mm, and then placed into a vacuum drier. Drying parameters: 100mb pressure, temperature 25
0C, 24 h. After this time, samples were weighted separately with precision to three decimal
places. To carry out studies of the kinetics of drug release 6 samples were prepared 5 samples
for testing the kinetics of release, and 1 sample for characterization under Scanning Electron
Microscopy - SEM. Gentamicin sulfate solution was prepared at a concentration of 50 mg /
ml using distilled water.
2.2.3. Soaking with gentamicin sulfate solution: Each sample was rinsed with distilled
water and then immerged into 3 ml of GS solution for 12 hours. After that time, soaked
electrospun fibers were removed from drug solution, then gently rinsed with PBS and dried
under vacum for 24 hours.
2.2.4. Drug releasing study:Kinetic of drug release researches was performed in a
simulated body fluids (PBS) at pH 7.4 and at 370C. Each sample after soaking in antibiotic
solution and drying was placed in a glass bottle filled with 3 ml of PBS in such way that the
largest area of contact between the sample and buffer was established. The samples were
incubated for 1, 2, 4, 8, 10, 24, 48, 72, 120, 168, 240 hours. After each PBS collecting,
exactly the same volume of fresh PBS was added.
2.2.5. GS concentration measurements: The amount of the GS released from the samples
was determinate under UV-VIS spectroscope. It is important to mention that it is not possible
to determine GS on spectrophotometer in pure form. In this case the procedure described by
M.R. Vitro [6] and H. Young [7] based on the use of OPA (o-phtaldialdehyde) reagent was
used. OPA reagent was obtained by mixing 2.5g of the OPA, 62.5ml of methanol and 3 ml of
2-mercaptoetanol with 560ml 0.04M solution of sodium tetraborate in distilled water 24 h
before measurement. Before measurements of the concentration of the GS in collected PBS
equal amounts of OPA reagent, GS solution in PBS and isopropanol are mixed and kept for
30 min at RT, which resulted in the reaction between OPA and amino groups of gentamicin
sulfate. The measurements of the GS concentration were performed on spectrophotometer at
wave length of 332 nm.
3. RESULTS AND DISCUSION
3.1. GS Release Profile
The gentamicin sulfate released from the samples was determined using UV-VIS
spectroscope. The amount of gentamicin sulfate released after 240 hours is shown in Figure 1.
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0 20 40 60 80 100 120 140 160 180 200 220 240 260
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
72
74
76
78
80
82
84
Cumulative % of GS released
Time [ h]
Cumulative % of GS released
Time [h] 
Figure 1. Total percentage of GS released, versus time
The results indicated that almost 80 % of the drug absorbed on the surface of the PLLA fibers
was released during first 10 h of the experiment. After this period, concentrations of GS in
PBS were under detection limit of the assay. Lucas-Girot et al. [5] indicated similar situation
with calcium carbonate materials soaked with GS where continuous release was observed
until 4 days of the experiment. By the end of the study only 40% of the soaked GS was
released at detectable levels.

3.2. Weight Measurements
Weight changes of the polymeric fibrous mats during whole study are presented in Fig. 2.
0
50
100
150
200
250
300
before GS soaking
after GS soaking
after GS released
Figure 2. Weight changes during experiment
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The difference in weight of the sample before and after soaking with the drug is the ratio of
weight of absorbed drug per unit mass of soaked material drug concentration in the material at
time 0. Average value of drug weight in the material is around 170% ± St. Div of initial
weight of material.
The results indicated that this method is suitable to reach high concentration of GS in poly (L-
lactide) (PLLA) fibrous and no significant difference in weight that samples before GS
soaking and after GS releasing can be found. This suggests that almost all drug was released.
Differences in value of soaked drug by samples can be explain by no uniform porosity of
fibers.
3.3. Surface Morphology Observations
In order to check whether the entire drug was released into the simulated body fluids scanning
electron microscopy observation of the surface of the electrospun poly(L-lactide) (PLLA)
fibrous mate were performed. The SEM images show changes in morphology of the surface
during whole experiment. Figure 3a shows sample before GS soaking. Sample immerged into
3 ml of GS solution for 12 hours and dried under vacum for 24 hours is shown in Figure 3b.
In this concentration almost whole surface of the sample is covered by the drug. However,
because of high surface tension, antibiotic did not immerged in deeper areas of the polymeric
mate. Figure 3c represents the sample after the GS releasing in to 3 ml of PBS for 240 hours.
It indicates that whole amount of the drug was released. However, on image with
magnification of 10 000 small amount of drug were still visible on fiber surafce.

Figure 3. Scanning electron microcopy (SEM) images: a) before GS soaking, b) after GS
soaking, c) after GS releasing; red arrows indicate GS and green arrows show deeper areas of
mate which are free of GS. The 3 different magnifications
(x2000, x5000, x10000 from left to right) were used for each sample
a a a
b
b
b
cc c
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3.4. FTIR Measurements
Fourier Transform Infrared spectroscopy (FTIR) measurements were performed to investigate
of each step of this experiment. FTIR spectra were shown in Figure 4. Spectrum for poly(L-
lactide) fibrous mat soaked with GS indicate additional peaks at 2888.2 cm-1, 1617.9 cm-1 and
1528.3 cm-1 which can be identified as peaks from amide and amino groups characteristic
for gentamicin sulfate. There were no significant differences observed in spectra of samples
before soaking and after release of the drug. However, sensitivity of FTIR measurement is not
suitable for investigation for residues amount of drug which was observed on SEM images.

Figure 4. FTIR spectra
CONCLUSIONS
In this study, poly(L-lactide) fibers soaked with GS was investigated as a drug delivery
system. The gentamicin sulfate release profile was performed 240 hours time period in
simulated body fluids. Results indicated that almost 80 % of the drug absorbed on the surface
of the PLLA fibers was released during first 10 h of the experiment. After this period, the
concentrations of the GS released were under the detection limit of the assay. The weight
measurements of the samples before soaking and after release suggest that the most of
absorbed drug was released. SEM investigations indicate that the almost whole surface of the
samples was covered by drug. However, because of high surface tension of the fibers
antibiotic did not immerged in deeper areas of the mat.
Feather study should be focused on improvement of the drug delivery system with respect to
soaking and concentration of the drug.
Acknowledgments: This work was supported by Grant No. PBZ/MNiSW-01/II/2007.
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5. A. Lucas-Girot, M.C. Verdier, O. Tribut, J.Ch. Sangleboeuf, H. Allain, H. Oudadesse, “Gentamicin-Loaded
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