development of chitosan nanoparticles for gene delivery using electrohydrodynamic spraying...
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Development of Chitosan Nanoparticles for Gene Delivery Using
Electrohydrodynamic Spraying Techniques
Orapan Paecharoenchaia, Tittaya Suksamranb, Tanasait Ngawhirunpatc, Theerasak Rojanaratad, Praneet Opanasopite
Faculty of Pharmacy, Silpakorn University, Nakhon Pathom 73000, Thailand
Keywords: Chitosan Nanoparticle; Gene Delivery; Electrospraying
Abstract. Chitosan nanoparticles were prepared by crosslinking chitosan (CS) with tripolyphosphate
(TPP) solution using electrohydrodynamic spraying technique. The effects of CS and TPP
concentration as well as electrical potential on particle size and shape were investigated. Appropriated
formulations for preparing nanoparticles were chosen to encapsulate DNA. In vitro evaluation of the
obtained nanoparticles as gene carrier such as entrapment efficiency and DNA release was performed.
The results showed that 2 mg/ml TPP was dropped at 10 kV into 1 mg/ml CS (MW 20 kDa (F1) and
200 kDa (F2)) yielded the spherical shape and small particles of 227.67 and 240.33 nm, respectively.
In DNA entrapment study, all formulations were tested by altering DNA loading to 10, 25 and 50 mg/g
of CS. The results revealed that F1 with initial DNA 10 mg/g of CS showed the highest entrapment
efficiency of 95.31%. While F2 with initial DNA of 25 mg/g of CS showed the highest entrapment
efficiency of 89.16%. The DNA release study from CS nanoparticles indicated that the increasing of
DNA amount slowed down the release rate. F1 and F2 with the initial DNA of 10 mg/g of CS had
faster release rate than those with 25 and 50 mg/g of CS. It can be concluded that F1 yielded the
nanoparticles with the smallest size, high DNA entrapment efficiency and enabled DNA sustained
release.
Introduction
One of the prerequisites for the successful gene therapy is the development of safe and efficient gene
delivery system. Non-viral vector has become the attractive alternatives to viral vectors due to severe
limitations of viral carriers such as possible toxicity, immunogenicity, mutagenesis and
carcinogenesis [1]. One of the most recently used non-viral vectors for gene delivery is chitosan, a
biodegradable polysaccharide obtained from N - deacetylation of chitin. Chitosan (CS) is soluble in
water at acidic pH due to the protonation of amino groups and insoluble in water at neutral or basic
pH. CS and its derivatives are preferable to be used as drug and gene delivery due to their properties
such as biocompatibility, biodegradability, non-toxicity [2]. Because of the amino groups of CS can
be protonated at acidic pH, thus CS becomes positive charge that enable to bind with negative charge
of DNA or siRNA through an electrostatic interaction to form complexes, therefore CS has a potential
as gene carrier [1]. In addition, many researchers have developed CS-based formulations by using the
ionic gelation technique with tripolyphospate (TPP) as crosslinking agent in order to prepare
nanoparticles [3-4]. Electrospraying, one of the electrohydrodynamic techniques, used for preparing
CS nanoparticles that are able to encapsulate protein and nucleic acid, etc.[5] Nanoparticles provide
the advantages as gene delivery such as enable to encapsulate with DNA and siRNA [1], protect
nucleic acid from enzymatic degradation and can be high uptaken by cells due to the small particle
size [6]. In this study, the CS nanoparticles were prepared by ionic gelation method with TPP and
using electrospraying process. The effects of various factors such as concentration of CS and TPP,
MW of CS, applied voltage and preparation method on particle size of CS nanoparticles were
Advanced Materials Research Vols. 194-196 (2011) pp 541-544Online available since 2011/Feb/21 at www.scientific.net© (2011) Trans Tech Publications, Switzerlanddoi:10.4028/www.scientific.net/AMR.194-196.541
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investigated. In addition, the abilities of nanoparticles in terms of DNA- entrapment efficiency, DNA
entrapment and cumulative DNA release were evaluated.
Materials and Methods
Chitosan with molecular weight 20 and 200 kDa and 87% degree of deacetylation were purchased
from Seafresh Chitosan Lab., Thailand. Aspartic acid was from Fluka, Switzerland. Chitosan
aspartate (CSA) was prepared by dissolving chitosan in aspartic acid solutions then used spray dried
method. Sodium tripolyphosphate (TPP) and Calf Thymus DNA were obtained from Sigma –
Aldrich, Germany. Other reagents were all of analytical grade.
CS nanoparticles were prepared by an ionic gelation method and using electrical spraying
technique according to a previous report procedure [7]. Briefly, CSA and TPP solution were prepared
by dissolving in distilled water to a concentration of 1-10 mg/ml for CSA and 0.2-2 mg/ml for TPP.
The instrument was settled up according to a scheme presented in Fig. 1. An upper phase solution
10 ml was filled in a glass syringe capped with 20 gauge blunt-end needle (an internal diameter of
needle is 0.9 mm) that was connected to negative electrode while a metal container containing a lower
phase solution in volume of 150 ml was connected to ground electrode. Lower phase solution was
stirred by using magnetic stirrer and distance between needle tips to the metal container was fixed at
30 cm. When applied the voltage (Gamma High voltage research, ORMOND BEACH) the upper
phase solution was extruded drop-wise through the needle into the lower phase solution. The
preparation methods of CS-TPP nanoparticles were done into two conditions; first, TPP was used as
upper phase while CS was lower phase and another one was prepared by inverting phases between CS
and TPP. Moreover, the applied voltage was varied from 8-18 kV in the present study. The resultant
particles were collected for size measurement. The appropriate formulations that could produce
nanoparticles were chosen from the first experiment in order to prepare DNA-loaded CS
nanoparticles. DNA was mixed with TPP solution and alterd the amount of DNA loading in three
levels (10, 25 and 50 mg per g of CS). To determine the amount of DNA-loaded in nanoparticles, a
centrifuge tube was weighed and filled the resultant nanoparticles then, they were centrifuged at
10,000 rpm/min 25 ºC for 10 min. The supernatant was drained and the solid particle was collected.
The dry weight of nanoparticles was calculated after lyophilized by using freeze-dryer (LABCONCO,
Freezone 2.5, USA). To determine DNA amount in the particles, 5 ml of 1N HCl was added into
100 mg of solid particles then they were sonicated until total particles were dissolved. The amount of
DNA in the obtained solution was determined using Gene Ray UV photometer (Biometra®
λ260/280 nm) at 260 nm. DNA entrapment efficiency was calculated by comparing with standard
curve. The percentage yield, DNA entrapment and percentage entrapment efficiency were calculated
according to method of Suksamran et al [7]. In vitro release study of DNA from nanoparticles was
performed by adding 1.2 ml of phosphate buffer saline (PBS) pH 7.4 into microcentrifuge tubes
containing DNA-loaded CS nanoparticles which accurately weighed 100 mg (n=3). All tubes were
shaken at 200 rpm 37±0.5ºC using shaking incubator (orbital Shaking Incubator Model: SI4) and
sample were collected at 0.25, 0.5, 1, 2, 4, 8, 20, 45, 72 hr. At the given time, the supernatant was
collected by centrifugation at 1,077 ×g for 15 min and fresh medium were refilled after each
sampling. To determine the amount of DNA released, the supernatant was performed by measuring
the absorbance at 260 nm compared the results with standard curve.
Fig. 1 Preparation of nanoparticles by using electrohydrodynamic spraying technique
542 Advanced Engineering Materials
Results and Discussion
Particle size of nanoparticles is an important factor affecting the epithelial tissue uptake and the
trafficking of nanoparticles within the cells. In addition, some research studies using the cell culture
technique stated that the gene transfection levels of nanoparticles with a smaller size was significantly
higher than the larger size [8]. This study was focused on various factors that might influence on
particle size of CS nanoparticles expected for gene delivery purpose. It was found that the higher the
CS concentration, the larger the particle size obtained. These were in accordance with a previously
reported [9] whereas TPP concentration did not influence on particle size. Effect of electrical
potential on particle size revealed that the particles with smallest size were resulted from the applied
voltage 18 kV (the highest voltage used in this study), however it could not be concluded that the
decreasing particle size caused by the increasing applied voltage. In the same way as the voltage, MW
of CS also influenced on particle size but how the MW affect on particle size was still unconcluded
from this experiment. However, when considered only the formulations that yielded nanoparticles; F1
and F2, it was found that F1(CS 20 kDa) was smaller than F2 (CS 200 kDa). These results were
corresponding with previous report results [10] that nanoparticles prepared from low MW CS were
smaller than high MW CS. Considering of the preparation method, results from phase inversion
studies indicated that particle size was smaller when using CS as a lower phase and TPP as an upper
phase. The formulations that could produce nanoparticles were provided in Table 1. The results
showed that when applied the voltage at 10 kV, CS as a lower phase and TPP as an upper phase (F1)
was used, the nanoparticles with the smallest size were obtained. In DNA entrapment study,
formulations F1, F2 and F3 were chosen for preparing DNA-loaded CS nanoparticles. The highest
percentage yield was obtained from F2 with initial DNA 50 mg/g CS.
Table 1. Formulations that yielded Chitosan - TPP nanoparticles
Formula Upper phase Lower phase Voltage (kV) Particle size (nm)
F0 TPP 2 mg/ml CS 20 kDa 1
mg/ml 0 220.30
F1 TPP 2 mg/ml CS 20 kDa 1
mg/ml 10 227.67
F2 TPP 2 mg/ml CS 200 kDa 1 mg/ml 10 240.33
F3 CS 20 kDa 1 mg/ml TPP 1 mg/ml 8 534.50
The DNA entrapment and percentage entrapment efficiency of all formulations were shown in
Fig. 2a-2c. It was found that F1 and F3 with initial DNA 10 mg/g CS showed the highest entrapment
efficiency, and the entrapment efficiency was decreased when initial DNA-loading increased. While
F2 showed the highest entrapment efficiency at initial DNA 25 mg/g CS.
Advanced Materials Research Vols. 194-196 543
Considering of DNA entrapment, F1 and F2 had the same pattern which the higher DNA
entrapment obtained from higher DNA-loading. The DNA release patterns of F1, F2 and F3
demonstrated that F1 and F2 with initial DNA of 10 mg/g CS showed the fastest release. In addition,
completely released (about 100%) of F1 and F2 occurred within 45 and 4 hr, respectively.The DNA
released from F1 and F2 with initial DNA 25 and 50 mg/g CS was slowly, and the cumulative released
within 192 hr of those formulations were incomplete as shown in Fig. 3a-3b. For F3, the fastest
released within 4hr occurred when initial DNA was 10 and 25 mg/g CS. When the initial DNA was
50 mg/g CS, the release rate of F3 was higher than F1 and F2. Moreover, the cumulative released of
F3 with initial DNA 50 mg/g CS within 68 hr was higher than F1 and F2 at the same period of time. It
could be concluded that F1 and F2 with initial DNA 25 and 50 mg/g CS and F3 with initial DNA
50 mg/g CS provided the sustained release pattern whereas immediate release occurred in all
formulations when initial DNA 10 mg/g CS.
Conclusion
In the present study, chitosan nanoparticles were successfully prepared from ionic gelation method by
crosslinked with tripolyphosphate and using electrical spraying process. The influences of various
factors on particle size were studied in order to obtained the particles in nanosize. For gene delivery
purpose, DNA was loaded into chitosan nanoparticles. The evaluations of DNA-loaded chitosan
nanoparticles in terms of entrapment efficiency, DNA entrapment and DNA release were performed.
The results indicated that some formulations were interesting to be developed as the controlled
delivery system of gene because of their abilities to produce nanoparticles with a small size, high
entrapment efficiency and enable the sustained release. Furthermore research studies about
transfection efficiency, safety and stability of these systems may be further investigated.
Acknowledgements
This work was financially supported by Commission of Higher Education (Thailand), the Thailand
Research Funds through the Golden Jubilee Ph.D. Program (Grant No.PHD/0092/2551) and the
Silpakorn University Research and Development Institute.
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544 Advanced Engineering Materials
Advanced Engineering Materials 10.4028/www.scientific.net/AMR.194-196 Development of Chitosan Nanoparticles for Gene Delivery Using Electrohydrodynamic Spraying
Techniques 10.4028/www.scientific.net/AMR.194-196.541
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