IIUM Research, Invention and Innovation Exhibition 2011‘Enhancing Quality Research and Innovation for Societal Development’

Farahidah Mohamed , Abd Almonem Doolaanea and Ahmad Fahmi Harun IsmailDepartment 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

ID: 300FABRICATION OF A GENE DELIVERY SYSTEM FROM A BIODEGRADABLE

POLYMER

RESULTS AND DISCUSSION

METHODS

INTRODUCTION

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 (10.25±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

ABSTRACT

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.

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.

Fabrication of pDNA-loaded PLGA microspheres:

PLGA 5% in DCM +

hydrophobic surfactants

pDNA in TE + hydrophilic surfactants

Primary Emulsion (W/O)

Aqueous PVA solution

Homogenisation 14500 RPM

1 min

Secondary Emulsion (W/O/W)

Homogenisation 14500 RPM

3 min

Aqueous PVA solution(Hardening Tank)

Solvent evaporationFreeze-drying

Centrifugation and washing

pDNA-Loaded PLGA Microspheres

1. Effect of different types of surfactants and surfactant blends on pDNA-loaded PLGA microspheres.

*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).

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.

Sample SURFACTANTS SURFACTANT BLEND* HLB PARTICLE SIZE (µm) ENCAPSULATION EFFICIENCY (%)

1 PVA (control) PVA (1%) - 10.37 ± 0.2 43.27 ± 10.87

2 Span88% span-8012% span-85

4 18.34 ± 0.10(S) 6.86 ± 3.89(S)

3 Span & Tween75% span-8025% tween-80

7 28.56 ± 0.85(S) 13.21 ± 8.03(S)

4 Span & Tween46% span-8054% tween-80

10 20.45 ± 2.68(S) 34.27 ± 8.30(NS)

5 Span & Tween14% span-8086% tween-80

13.5 17.03 ± 0.16(S) 19.09 ± 3.05(S)

6 Tween41% tween-8059% 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-8062% TX100

10 29.88 ± 0.49(S) 38.44 ± 3.98(NS)

11 TX100 & CTAB50% CTAB50% TX100

11.75 1.72 ± 0.01(S) 9.14 ± 5.97(S)

Table 1.1. SEM images for pDNA-loaded PLGA microspheres with different surfactants.

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:

Sample PVA CONCENTRATION SURFACTANT PARTICLE SIZE (µm) ENCAPSULATION EFFICIENCY (%)

1 1% w/v TX100 10.25 ± 0.16 34.19 ± 7.202 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 (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.

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.

CONCLUSIONSurfactants 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.

REFERENCES1. 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.

Table 2.1. Effect of PVA concentration on the particle size and encapsulation efficiency of the pDNA-loaded PLGA microspheres.

1%PVASpan&Tween HLB=4Span&Tween HLB=10Span&Tween HLB=16

TX100SDSCTABTX100 & CTAB