FABRICATION OF A GENE DELIVERY SYSTEM
FROM A BIODEGRADABLE POLYMER
ID: 300
Farahidah Mohamed , Abd Almonem Doolaanea and Ahmad Fahmi Harun Ismail
Department of Pharmaceutical Technology, Faculty of Pharmacy, International Islamic University of Malaysia,
25200 Kuantan Campus, Malaysia.
Phone: 09-571-6400 Fax: 09-571-6775 E-mail: farahidah@iiu.edu.my
ABSTRACT
Table 1.2. Effect of different types of surfactants and surfactant blends on the particle size and encapsulation
efficiency of the pDNA-loaded PLGA microspheres. Size obtained was statistically compared against control.
Biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres is a promising gene carrier
system to deliver the gene in gene therapy. This study describes fabrication and characterization of
plasmid DNA-loaded PLGA microspheres using a versatile double-emulsion (w/o/w) solventevaporation method.
For microsphere fabrication, plasmid DNA in TE buffer was added to PLGA solution, previously
dissolved in DCM, and was homogenized to form the primary water-in-oil (w/o) emulsion. This w/o
emulsion was immediately injected into 1% aqueous PVA and homogenized to form the secondary
water-in-oil-in-water (w/o/w) emulsion. The w/o/w emulsion was then transferred to a continuously
stirred hardening tank of 1% aqueous PVA and the stirring was continued for 2 hours to allow
complete evaporation of DCM. The hardened microspheres were collected by centrifugation, washed
and freeze-dried.
Several parameters have been investigated including PVA concentration, different types of
surfactants and surfactant blends.
Resultant microspheres were characterized for size distribution and external morphology by laser
sizer and scanning electron microscopy, respectively. Encapsulation efficiency was also calculated
and the DNA was quantified by UV absorbance (NanoQuant). It was found that, increasing the PVA
concentration from 1% w/v to 5% w/v reduced the mean particle size from (10.25±0.16) µm to (3.39 ±
0.01) µm. These sizes were also evident by the microimages that depicted smooth surfaces of
microspheres yielded for the range of PVA concentration. This research is still ongoing and future
aims include transfection on neuro cell line to see feasibility of using this carrier system to deliver
relevant gene to treat neurodegenerative diseases at molecular level.
OBJECTIVES
To study the effect of different type of surfactants and surfactant blends on pDNA-loaded PLGA
microspheres.
To investigate the effect of polyvinyl alcohol (PVA) concentration on the particle size and
encapsulation efficiency of the microspheres.
INTRODUCTION
Microencapsulation is a promising gene delivery system and has made significant improvements last
years. DNA encapsulated in microspheres can be protected from nuclease degradation, delivered to
specific sites and sustained release can be achieved without the need for frequent administration1.
Polylactide (PLA) and poly (lactide-co-glycolide) (PLGA) are currently the most commonly used
polymers as they are biodegradable, biocompatible and nontoxic2.
Microsphere characteristics like particle size and surface properties are important to achieve
successful delivery system. For example, rapid cellular uptake of DNA fabricated in less than 10 μm
microparticles aids in the escape from interstitial nuclease-mediated degradation and directed to
tissues depending on injection route, size, and surface characteristics3. Good encapsulation of the
pDNA is requested for effective delivery and to avoid loss of DNA during fabrication process.
Surfactants can affect microsphere properties like surface morphology4, DNA loading4 and burst
release5.
In this study, different PVA concentrations (1-5 % w/v) and five types of surfactants (Span, Tween,
Triton X100, SDS and CTAB) were employed in fabrication of pDNA-loaded PLGA microspheres to
investigate their effect on microsphere characteristics including particle size, encapsulation efficiency
and surface morphology.
METHODS
Fabrication of pDNA-loaded PLGA microspheres:
PLGA 5% in
DCM +
hydrophobic
surfactants
pDNA in TE
+
hydrophilic
surfactants
Sample
SURFACTANTS
SURFACTANT BLEND*
1
PVA (control)
PVA (1%)
2
Span
3
Span & Tween
4
Span & Tween
88% span-80
12% span-85
75% span-80
25% tween-80
46% span-80
54% tween-80
14% span-80
86% tween-80
41% tween-80
59% tween-20
HLB
PARTICLE SIZE (µm)
ENCAPSULATION EFFICIENCY (%)
-
10.37 ± 0.2
43.27 ± 10.87
4
18.34 ± 0.10(S)
6.86 ± 3.89(S)
7
28.56 ± 0.85(S)
13.21 ± 8.03(S)
10
20.45 ± 2.68(S)
34.27 ± 8.30(NS)
13.5
17.03 ± 0.16(S)
19.09 ± 3.05(S)
16
12.45 ± 0.03(S)
28.09 ± 6.91(NS)
5
Span & Tween
6
Tween
7
TX100**
100% TX100
13.5
10.25 ± 0.16(NS)
34.19 ± 7.20(NS)
8
9
SDS***
CTAB****
40
10
9.27 ± 0.05(S)
2.23 ± 0.01(S)
46.14 ± 8.72(NS)
10.78 ± 0.82(S)
10
TX100 & Span
10
29.88 ± 0.49(S)
38.44 ± 3.98(NS)
11
TX100 & CTAB
100% SDS
100% CTAB
38% span-80
62% TX100
50% CTAB
50% TX100
11.75
1.72 ± 0.01(S)
9.14 ± 5.97(S)
*The amount of total surfactants is 1% of the primary emulsion except for sample No.11 where each surfactant is 1% of the primary
emulsion.
**TX100: Triton X100. ***SDS: Sodium Dodecyl Sulphate.. ****CTAB: Cetyl Trimethyl Ammonium Bromide.
(S): Statistically significant (P<0.05). (NS): Statistically not significant (P≥0.05).
1.1. Microsphere particle size: Table 1.2 clearly shows that the type of surfactant and the
surfactant blends can affect the microsphere particle size. Comparing the particle size with the
control (1%PVA), it seems that Span/ Span-Tween blends and CTAB, increased and strongly
reduced, respectively the particle size. In contrast, TX, SDS and Tween alone did not statistically
affect the particle size. For sample 3, 4 and 5, it shows that when the span fraction was increased
(decrease in HLB), the particle size tend to increase. These may be due to different stabilizing
mechanism of different surfactants. For example, for span groups, they stabilizes W/O emulsions
and hence tend to make the oil as a continuous phase that predispose the droplets in the
secondary W/O/W emulsion to merge. CTAB, a positively charged surfactant, may form a complex
with the negatively charged PLGA on the water-oil surface to stabilize the primary emulsion, and
also can alter the surface of the droplets in the secondary emulsion to become positively charged
leading to a more stable secondary emulsion by means of electrostatic repulsive effect. These
effects of CTAB lead to smaller droplets and thus smaller microspheres.
1.2. Microsphere encapsulation efficiency: Incorporating Span in the microspheres reduced the
EE. This may be explained by the formation of inverse micelles which transfer water and water
soluble plasmid through the oil layer. For Span-Tween blends, the highest EE found is that of the
HLB=10 blend. This complies with the primary emulsion stability which showed that HLB=10
produced the most stable emulsion (data not shown). No significant difference appeared with
TX100, Tween (HLB=16) or SDS (HLB=40) which are very hydrophilic surfactants and may behave
like PVA. Although CTAB may stabilize both primary and secondary emulsion, it can form complex
with the negatively charged plasmid and the resultant complex has more oil solubility than the
plasmid and then facilitate the escape through oil layer. Also, CTAB strongly reduced the particle
size resulting in increased surface area which facilitates permeability of the plasmid to the outer
W2 phase.
1.3. Surface morphology: All surfactants and surfactant blends in this study produced smooth
surface microspheres.
2. Effect of PVA concentration on pDNA-loaded PLGA microspheres:
Homogenisation
14500 RPM
1 min
Aqueous PVA
solution
Table 2.1. Effect of PVA concentration on the particle size and encapsulation efficiency of the pDNA-loaded PLGA
microspheres.
Homogenisation
14500 RPM
3 min
Primary Emulsion
(W/O)
Freeze-drying
Centrifugation and
washing
DCM
DCM
DCM
Secondary Emulsion
(W/O/W)
Sample
PVA CONCENTRATION
SURFACTANT
PARTICLE SIZE (µm)
1
2
3
4
5
1% w/v
2% w/v
3% w/v
4% w/v
5% w/v
TX100
TX100
TX100
TX100
TX100
10.25 ± 0.16
10.35 ± 0.15(NS)
7.77 ± 0.03(S)
3.65 ± 0.49(S)
3.39 ± 0.01(S)
ENCAPSULATION
EFFICIENCY (%)
34.19 ± 7.20
20.03 ± 0.53(S)
17.54 ± 3.89(S)
14.90 ± 1.47(S)
9.16 ± 1.28(S)
(S): Statistically significant (P<0.05)
compared to sample No.1.
(NS): Statistically not significant
(P≥0.05) compared to sample No.1.
Table 2.2. SEM images blank PLGA microspheres with different PVA concentration.
Solvent
evaporation
Aqueous PVA solution
(Hardening Tank)
pDNA-Loaded PLGA
Microspheres
RESULTS AND DISCUSSION
1. Effect of different types of surfactants and surfactant blends on pDNA-loaded PLGA
microspheres.
PVA 1% w/v
PVA 3% w/v
PVA 5% w/v
It is clear that increasing the PVA concentration reduced the particle size. This may be due to
increasing the viscosity of W2 phase and thus hindering emulsion droplets from combining
together. Increasing the surface area of the smaller particles may play a role in the reduction of
EE when increasing the PVA concentration. PVA concentration did not affect the surface
morphology where 1, 3 and 5% w/v PVA produced smooth microspheres.
Table 1.1. SEM images for pDNA-loaded PLGA microspheres with different surfactants.
CONCLUSION
Span&Tween HLB=16
Span&Tween HLB=10
Span&Tween HLB=4
1%PVA
Surfactants and surfactant blends can modify the microsphere characteristics including particle
size, encapsulation efficiency and surface morphology. PVA concentration plays an important role
in determining microsphere particle size. Understanding the role of each parameter can aid in
designing microspheres with specific properties.
REFERENCES
TX100 & CTAB
CTAB
SDS
TX100
1. William C. Heiser (ed.), Nonviral gene transfer techniques, vol. 1, Gene delivery to mammalian cells, (New Jersey: Humana Press Inc., 2004), 157-158.
2. Susanna, W.P. & Yon, R. (eds.), Biopharmaceutical drug design and development (2nd edn), (Humana Press, 2008), 308.
3. Mansoor, M.Amiji (ed.), Polymeric gene delivery: Principles and applications, (CRC Press LLC, 2005), 531-533.
4. Mohamed, F. and C. F. van der Walle (2006). "PLGA microcapsules with novel dimpled surfaces for pulmonary delivery of DNA." Int J Pharm 311(1-2): 97-107.
5. Bouissou, C., J. J. Rouse, et al. (2006). "The influence of surfactant on PLGA microsphere glass transition and water sorption: remodeling the surface morphology to
attenuate the burst release." Pharm Res 23(6): 1295-1305.
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