Tailored PLGA nanoparticles emulsified with TPGS for controlled ocular drug delivery Musarrat Husain Warsi1,, Mohammad Yusuf1, Majed Al-Robaian1, Mohammad Akhlaquer Rahman1, Gaurav K Jain2 1College
of Pharmacy, Taif University, Taif, KSA 2Nanomedicine Lab, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India
OCULAR SAFETY EVALUATION
CHARACTERIZATION Particle Size Measurement of particle size, zeta potential and polydispersity of nanoparticles was done using Zetasizer (Nano ZS, Malvern Instruments, Malvern, UK), which is based on the principle of dynamic light scattering (DLS).
✓0.1N NaOH: Positive control ✓ 0.9% NaCl: Negative control Response
A combination of Drug:PLGA ratio of 1:15 and Vit E TPGS ( 0.5% w/v) as emulsifying agents favoured the production of smaller nanoparticles with mean size of 225.8±3.8 nm with PDI of 0.029±0.008.
Particle Morphology Morphological evaluation was determined by taking SEM and TEM images of drug nanoparticles using a ZEISS EVO Serie112s EVO 50 (Carl Zeiss International, Germany) microscope operating at an accelerating voltage of 13.52 kV under high vacuum. SEM MICROGRAPH
PREPARATION AND OPTIMIZATION OF NANOPARTICLES PREPARATION: SOLVENT EVAPORATION METHOD
SEM images showed that the particles have moderate uniformity. NPs with narrow size distribution could be achieved with appropriate formulation process. Aggregation was also observed, TPGS acts as surfactant in particles fabrication and the use of significant quantity of surfactant in formulation has been found to cause particles aggregation.
TPGS stabilized, Betoxolol loaded PLGA nanoparticles were produced by the emulsification technique
ORGANIC PHASE PLGA dissolved in acetone
Estimation of the particle size from the TEM images was in good agreement with the values obtained by laser light scattering method.
LYPPHILIZATION
Ultrsonication
AQUEOUS PHASE 0.5% TPGS solution containing model drug
LYOPHILIZED Betaxolol NPs
OPTIMIZATION OF PLGA CONC.
OPTIMIZATION OF pH VALUE
OPTIMIZED FORMULATIO N
❖ Organic solvent→ Acetone ❖ PLGA concentration → 2%w/v ❖ pH → 5.5 ❖ Drug : PLGA ratio → 1:15 ❖ TPGS conc. → 0.5% w/v ❖ W/O Phase → 5:1
TEM MICROGRAPH
BNP1 BNP2 BNP3 BNP4 BNP5
Particle Size (nm) ±SE 273.1±4.8 238.3±3.6 228.5±3.2 225.8±3.8 256.1±4.1
Polydispersity index (nm) ±SE 0.122±0.032 0.104±0.026 0.071±0.013 0.029±0.008 0.095±0.011
Drug entrapment (%) ±SE 38.8±1.1 41.8±1.4 51.3±1.6 59.4±1.9 54.4±1.7
Response Score
0.5
2.0
5.0
Cumulative Score
Irritation Potential
No effect
0
0
0
0.0 – 0.9
None
Hyperemia
5
3
1
1.0 – 4.9
Slight
Haemorrhage
7
5
3
5.0 – 8.9
Moderate
Clotting
9
7
5
9.0 – 21.0
Strong
▪ In HET-CAM test: ➢ Cumulative score, 0.1 M NaOH =18.98 (Strong irritant) ➢ Cumulative score , saline solution = 0 (Non-irritant) ➢ Cumulative scoreDrug NPs (TPGS) = 0.33 (Non-irritant) ➢ Cumulative score Drug solution = 0 (Non-irritant) ▪ Histological study of goat cornea confirmed the presence of normal ocular structures, with cells maintaining a normal morphology in both, control and nanoformulation treated samples. ▪On the basis of the results obtained in the above tests performed, the developed nanoparticles under study were found to be devoid of irritation potential and safe for ocular administration in vivo.
CONCLUSION The optimized Drug NPs (TPGS), fabricated by modified emulsification/evaporation method were found to possess size in range of 225.8±3.8nm and narrow size distribution. Also, a much higher encapsulation efficiency of drug was achieved in comparison to methods reported in literature that use traditional stabilizers. Drug loaded NPs (TPGS) were found to be non-irritant in ocular safety studies, and were thus safe for ocular instillation. In vitro transcorneal permeability studies revealed that the bioavailability of model drug was significantly higher when administered in the form of formulated nanoparticles as compared to solution form. Thus, as hypothesised, the results of physico-chemical characterization together with in vitro studies indicate that the PLGA nanoparticles fabricated using TPGS as stabilizer are efficient carriers for ocular delivery of betaxolol for effective management of glaucoma. Further in vivo studies data, finally will reveal the future potential of this developed system.
REFERENCES
The transcorneal permeation study was carried out using standard Franz diffusion cells. Fresh whole eyeballs of goats were obtained from a local slaughter house and transported to the laboratory in cold condition in normal saline. Cornea was isolated and mounted on the diffusion cell. The receiver compartment of diffusion cell was filled with PBS, pH 7.4 as transport medium and nanoparticles suspended in normal saline were placed in the donor compartment. The perfusate was collected at periodic time intervals for up to 4 hours, filtered through 0.45-μm membrane and analyzed for drug content by UPLC method. The cumulative amount of drug permeated per unit of goat cornea surface area, Qt/S (S=0.75 cm2) was plotted as a function of time (h).
1. Warsi, M.H., Anwar, M., Garg, V., Jain, G.K., Talegaonkar, S., Ahmad, F.J., Khar, R.K. Dorzolamide-loaded PLGA/vitamin E TPGS nanoparticles for glaucoma therapy: Pharmacoscintigraphy study and evaluation of extended ocular hypotensive effect in rabbits. Colloids Surf B Biointerfaces. 1;122 (2014) 423-431. 2. Gupta, H., Aqil, M., Khar, R.K., Ali, A., Bhatnagar, A., Mittal, G. Sparfloxacin-loaded PLGA nanoparticles for sustained ocular drug delivery. Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 324–333. 3. Mu, L., Feng, S.S. Vitamin E TPGS used as emulsifier in the solvent evaporation /extraction technique for fabrication of polymeric nanospheres for controlled release of paclitaxel (TaxolÒ). J Controll. Release 80 (2002) 129–144. 4. Peltonen, L., Aitta, J., Hyvönen, S., Karjalainen, M., Hirvonen, J. Improved Entrapment Efficiency of Hydrophilic Drug Substance During Nanoprecipitation of Poly(l)lactide Nanoparticles. AAPS PharmSciTech 5(2004) 1-6.
S.No.
SAMPLE
ACKNOWLEDGEMENTS
Papp (cm/sec) × 10-6
The authors are thankful to Mohammad Anwar, Jamia Hamdard, for helping during this work. DZA SOL
2.02±0.25
OPTIMIZATION OF DRUG:POLYMER RATIO Drug to Polymer ratio 1:2 1:5 1:10 1:15 1:20
Time (min)
TRANSCORNEAL PERMEATION STUDIES
1.
Batch
HET - CAM
Ocular drug delivery is one of the most challenging endeavour faced by the formulation scientist. As being the vital organ of vision, eye is equipped with urbane anatomic structures and shielding mechanisms to preserve the highly regulated and confined milieu for its function. Due to these precise barricades the bioavailability of ocular drugs after topical instillation is quite poor. For effective handling of ocular ailments need to overcome these barriers. For the same, an optimum ocular delivery system should be design, that can fulfill the various attributes like to meet patient compliance, low dose frequency, well tolerated without causing blurring or irritation, and finally should be effective with increased permeability & ocular bioavailability. Formulation of polymeric nanoparticles as ocular drug delivery systems can meet the above prerequisite in a large extent. In present research, polymeric nanoparticles were produced by the emulsification technique. There are various fabrication parameters which affect the nature of the nanoparticles. Among them, the role of an emulsifier is an important one. Emulsifier stabilises the dispersed-phase droplets formed during emulsification, inhibits coalescence of droplets and determines the particle size, size distribution, the morphological properties as well as the release properties of the nanoparticles formed. Traditional and most popular emulsifier is poly(vinyl alcohol) (PVA), the use of which is plagued by various drawbacks such as difficulty in complete removal from formulated nanoparticles following the fabrication process. Hence, the present research was focused at exploring the emulsifier, D-α-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS, or TPGS) as an alternative to the traditional PVA for fabrication of PLGA nanoparticles as an ocular drug delivery system. TPGS presents several desirable attributes as a PVA substitute. Along with its nature as surfactant & stabilizer, it has also been reported to inhibit efflux transporters localized in the eye. These inherent qualities of TPGS in conjunction were hypothesized to facilitate the production of stable PLGA nanoparticles with the ability to enhance ocular bioavailability of entrapped drug betaxolol for effective management of glaucoma.
Lack of any ocular adverse effects is an essential requirement of a system intended for ocular use and is an essential prerequisite to any in vivo testing. Ocular irritation of the developed formulations was checked by HET-CAM test and post formulation treatment histopathological evaluation of goat corneas. ICCVAM (Interagency Coordinating Committee on Validation of Alternative Methods) recommended test method protocols were followed for performing the two tests. HISTOPATHOLOGICAL EVALUATION
INTRODUCTION
Zeta potential (mV) ±SE -21.2±1.2 -20.6±1.4 -18.2±0.8 -19.2±0.9 -19.7±1.1
2.
3.
DZA NP (PVA)
DZA NP (TPGS)
2.97±0.67
Papp of DZA NPs(TPGS) was found to be 2.5 times higher than DZA SOL
5.14±0.45
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