Farahidah Mohamed , Abd Almonem Doolaanea and Ahmad Fahmi Harun Ismail

1
ID 300 FABRICATION OF A GENE DELIVERY SYSTEM FROM A BIODEGRADABLE POLYMER
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 [phone] E-mail
farahidah_at_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)
solvent-evaporation 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
(10.250.16) 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.
Sample SURFACTANTS SURFACTANT BLEND HLB PARTICLE SIZE (µm) ENCAPSULATION EFFICIENCY ()
1 PVA (control) PVA (1) - 10.37 0.2 43.27 10.87
2 Span 88 span-80 12 span-85 4 18.34 0.10(S) 6.86 3.89(S)
3 Span Tween 75 span-80 25 tween-80 7 28.56 0.85(S) 13.21 8.03(S)
4 Span Tween 46 span-80 54 tween-80 10 20.45 2.68(S) 34.27 8.30(NS)
5 Span Tween 14 span-80 86 tween-80 13.5 17.03 0.16(S) 19.09 3.05(S)
6 Tween 41 tween-80 59 tween-20 16 12.45 0.03(S) 28.09 6.91(NS)
7 TX100 100 TX100 13.5 10.25 0.16(NS) 34.19 7.20(NS)
8 SDS 100 SDS 40 9.27 0.05(S) 46.14 8.72(NS)
9 CTAB 100 CTAB 10 2.23 0.01(S) 10.78 0.82(S)
10 TX100 Span 38 span-80 62 TX100 10 29.88 0.49(S) 38.44 3.98(NS)
11 TX100 CTAB 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
(Plt0.05). (NS) Statistically not significant
(P0.05).
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.

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 (1PVA), 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 HLB10 blend.
This complies with the primary emulsion stability
which showed that HLB10 produced the most stable
emulsion (data not shown). No significant
difference appeared with TX100, Tween (HLB16) or
SDS (HLB40) 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.
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
2. Effect of PVA concentration on pDNA-loaded
PLGA microspheres
Table 2.1. Effect of PVA concentration on the
particle size and encapsulation efficiency of the
pDNA-loaded PLGA microspheres.
Sample PVA CONCENTRATION SURFACTANT PARTICLE SIZE (µm) ENCAPSULATION EFFICIENCY ()
1 1 w/v TX100 10.25 0.16 34.19 7.20
2 2 w/v TX100 10.35 0.15(NS) 20.03 0.53(S)
3 3 w/v TX100 7.77 0.03(S) 17.54 3.89(S)
4 4 w/v TX100 3.65 0.49(S) 14.90 1.47(S)
5 5 w/v TX100 3.39 0.01(S) 9.16 1.28(S)
(S) Statistically significant (Plt0.05) compared
to sample No.1. (NS) Statistically not
significant (P0.05) compared to sample No.1.
Table 2.2. SEM images blank PLGA microspheres
with different PVA concentration.
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 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
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.