Synthesis and Characterization of Drug Loaded Nanoparticles using PEO-PPO-PEO Triblock

Copolymers

Presented by :

Vishal J. Patel

Guided by :

Dr.Rakesh K. Sharma

PEO-PPO-PEO Tri-block Copolymers Pluronic® Polymers (BASF Product)

Other Trade names : Poloxamers, Symperonics

• Nonionic surfactant available in varieties of mol.wt. with change in PO/EO

ratios/lengths of the two components.

• The most important characteristic property is the temperature-dependent

micellization.

Bahadur, P., Current Science, 80 (2001)1002-1007

PEO-PPO-PEO Triblock Copolymers

Pluronics as Nano-sized Drug Carriers

Work Mechanism : Encapsulation of drugs into Pluronic micelles

Pluronics are FDA approved and listed in US and British Pharmacopoeia

Kabanov, A.V. et. al, FEBS Lett., 258 (1989) 343

Kabanova, A. V. and Batrakova, E. V., Journal of Cont. Release, 130 (2008) 98–106

The overall aim of this study was to evaluate the PEO-PPO-PEO triblock

copolymers (Pluronic® Polymers) as the best candidate for encapsulation of

two imporatant drug, Curcumin and Lamotrigene for safer drug delivery

applications.

Curcumin-loaded nanoparticles using Pluronic® Polymers (F127, F68, P123,

L64) were prepared by thin film hydration method and characterized by

various techniques i.e. DLS, SEM, UV-VIS, FT-IR, XRD & DSC analysis.

In vitro release study of Curcumin drug from Pluronic micelles was shown

for sustainable release.

Lamotrigine-loaded nanoparticles using Pluronic® Polymers (F127 & F68)

were prepared also by thin film hydration method and characterized by

methods i.e. SEM, UV-VIS, FT-IR, & TGA analysis.

Interaction and thermodynamics parameters of both the drugs with Pluronic®

Polymers are discussed. 4

Present Study

Curcumin Loaded Nanoparticles (CURNPs)

Curcumin- The Indian Solid Gold

It is a polyphenolic

compound isolated from

rhizome of perennial herb,

turmeric (Curcuma longa).

It exhibits Keto - enol

tautomerism

Curcumin has been shown to exhibit antioxidant, anti-inflammatory,

anti-microbial. and anti-carcinogenic activities.

Anand, P., Kunnumakkara, A.B., Newman, R.A. and Aggrawal, B.B., Molecular Pharmaceutics, 130

(2008) 98–106

Materials

Pluronic® polymers was purchased from Sigma-Aldrich and used as received.

Curcumin drug was obtained from Loba Chemie and used with further purifications.

Preparation of Curcumin loaded Nanoparticles (CURNPS)

By Thin-Film Hydration method

Characterization of Curcumin loaded Nanoparticles (CURNPS)

In vitro release study of CURNPS

Two phase release profile was observed.

• Rapid release in first stage followed by sustained and slow release over o

prolonged time of 10 days.

• Pluronic F127 release drug more slowly than F68

1st Conclusion

• Curcumin-loaded Nanoparticles were successfully prepared

without use of any cryoprotectants.

• The Properties and interaction of curcumin-loaded

nanoparticles were successfully studied through UV-Vis, DLS,

SEM, FT-IR, XRD and DSC techniques.

• Rapid release in first stage followed by sustained and slow

release over o prolonged time of 10 days were found in

CURNPs.

• Pluronic® polymer has good candidate for Drug delivery

applications for Curcumin.

Lamotrigine Loaded Nanoparticles (LamoPNPs)

Lamotrigine Drug

Lamotrigine is a substituted asymmetric

triazine.

It is a white to pale cream colored powder.

It is slightly soluble in ethanol and

chloroform, and very slightly soluble in water.

The pKa of lamotrigine at 298K is 5.7.

The chemical name for lamotrigine is 3,5-

diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine.

Molecular weight: 256.1 g/mol Structure of Lamotrigine

No. Formulation Code Ratio of polymer and drug

Used polymer

1 LamoPNP1 1:10 F68

2 LamoPNP2 1:30 F68

3 LamoPNP3 1:50 F68

4 LamoPNP4 1:10 F127

5 LamoPNP5 1:30 F127

6 LamoPNP6 1:50 F127

Lamotrigine-loaded Pluronic nanoparticles (LamoPNP) formulations

Lamotrigine-loaded nanoparticles (LamoPNPs) were prepared by thin-film hydration

method.

Stock solutions of Lamotrigine and Pluronic® Polymers were prepared in methanol and

chloroform respectively.

Required amount of stock solutions according to drug–polymer ratios (mentioned in

formulation table) was transferred to separate RBFs.

The solvent was completely evaporated to obtain drug-containing solid polymer film

The residual organic solvent remaining in the solid was removed by keeping them in

vacuum oven at room temperature for overnight.

After that, the solid film was rehydrated in PBS (pH 7.0) (pre-warmed at 370C) by

extensive vortexing to prepare drug-loaded micelles.

Non-encapsulatedcurcumin drug was separated by centrifugation of the micelle

suspension at 5000 rpm for 10 min and filtration.

Lyophilized formulation was obtained by freeze-drying of Lamotrigine-loaded NPs in a

freeze dryer.

NPs samples were rapidly frozen by liquid nitrogen and attached to the freeze dryer.

During freeze drying, the chamber pressure was maintained at 0.035 mbar and

temperature was -55°C.

Finally we get samples of LamoPNPs, which one characterized to study the interaction

between drugs & copolymer F127.

Preparation of LamoPNPs

Lamotrigine Loaded Nanoparticles (LamoPNPs)

F127 1:10 F127 1:30 F127 1:50

F68 1:10 F68 1:30 F68 1:50

Calibration Curve of Lamotrigine

0.1 0.2 0.3 0.4 0.5 0.6 0.7

0.0

0.5

1.0

1.5

2.0

2.5

3.0

200 220 240 260 280 300 320 340 360 380

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Ab

so

rba

nc

e,

a.u

.

Wavelength, nm

1%

2%

3%

4%

5%

6%

7%

Concentration[mM]

Ab

so

rba

nc

e,

a.u

.

Equation y = a +

Adj. R-Squ 0.9968

Value Standard E

B Intercep -0.210 0.04475

B Slope 4.3442 0.10007

Blank experiments, without copolymer, were done to determine the solubility of

Lamotrigine in water. The amount of drug solubilized was determined by measuring

absorbance at 219 nm.

Calibration plot of Lamotrigine Drug

E. Perucca, Extended-release formulations of antiepileptic drugs: rationale and

comparative value, Epilepsy Curr. 9 (6) (2009) 153–157.

Concentration of Lamotrigine loaded NPs

Different formulations containing Drug

polymer ratio (w/w)

Drug: polymer ratio Nanoparticles yield

LamoPNPs 1 1 : 10 47.72

LamoPNPs 2 1 : 30 34.51

LamoPNPs 3 1 : 50 47.84

LamoPNPs 4 1 : 10 47.27

LamoPNPs 5 1 : 30 34.19

LamoPNPs 6 1 : 50 49.90

Drug loading, Entrapment efficiency and Nanoparticles yield of LamoPNPs.

Nanoparticle Name Drug: polymer ratio Absorbance at

219

Concentration

LamoPNPs F127 1:10 1.0809 0.3003

LamoPNPs F127 1:30 1.2138 0.3239

LamoPNPs F127 1:50 1.4228 0.3751

LamoPNPs F68 1:10 1.3975 0.3692

LamoPNPs F68 1:30 0.4857 0.1585

LAmoPNPs F68 1:50 0.9796 0.2781

Methods used for characterizing LamoPNPs are ;

1.Scanning Electron Microscopy (SEM)

2.Ultraviolet Spectroscopy (UV-VIS)

3.Fourier Transform-Infrared Spectroscopy (FT-IR)

4.Thermal Gravimetric Analysis (TGA)

Characterizations of LamoPNPs

SEM Analysis

Lamotrigine

Crystalline nature

Amorphous nature

Polymer

Amorphous nature

F127 1:50

UV-VIS

UV-Visible absorption spectra Pluronic

F127 and LamoPNP F127

200 220 240 260 280 300 320 340 360 380 400

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Wavelength, nm

Ab

so

rb

an

ce

, a

.u.

F127110

F127130

F127150

PolyF127

200 220 240 260 280 300 320 340 360 380 400

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Ab

so

rb

an

ce

, a

.u.

Wavelength, nm

F68110

F68130

F68150

PolyF68

UV-Visible absorption spectra Pluronic

F68 and LamoPNP F68

Higuchi and Connors 1965, Ahuja et al.2007, Yamaamoto et al. 2000; ahuja et al. 2007

Ruan et al. 2005.

FT-IR

FT-IR Spectra of (a) Lamotrigine, (b) Polymer F127, (c) Lamotrigine-loaded Nanoparticles

(A) Lamotriginr drug shows -N=N- stretching

at 1640 cm-1, -N-H stretching at 3500-3200

cm-1, C-Cl stretching at 800 cm-1, Aromatic

1660-1580 cm-1.

(B) Pluronic F127 showed two major peaks at

1101 cm-1of – C – O stretching and 2882 cm-1

of – C – H stretching.

(C) In LamoPNPs, three major peaks were at

C-Cl stretching at 800 Cm-1 , -C-O stretching

at 1090 cm-1 , -C-H stretching at 2890 cm-1.

aromatic 1660-1580 cm-1 .

The FT-IR spectra also confirmed the

interaction of Lamotrigine with F127 micelles

and identified the formation of nanoparticles

between them.

4000 3500 3000 2500 2000 1500 1000 5000

20

40

60

80

100

120

140

160

180

200

Tra

ns

mit

an

ce

(%

)

Wawenumber(cm-1)

(a)

(b)

(c)

TGA

TGA thermograms of (a) Lamotrigine (b) Plu F127,

and (c) LamoPNPs F127.

TGA thermogram of pure

Lamotrigine shows weight loss from

217°C - 670°C and Pluronic®F127

shows weight loss from 166°C - 475°C.

A fast process of weight loss

appeared in the TGA curve of

Lamotriginne loaded nanoparticles

(LamoPNPs) i.e. from 180°C - 475°C

respectively.

These results showed some decrease

of thermal stability for Lamotrigine

loaded nanoparticles relative to the pure

Lamotrigine drug.

100 200 300 400 500 600 700

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

We

igh

t (m

g)

Temp [C0]

Lamotrigine

PolyF127

NPsF127

2nd Conclusion

• Lamotrigine-loaded Nanoparticles were successfully prepared

without use of any cryoprotectants.

• The Properties and interaction of Lamotrigine-loaded

nanoparticles were successfully studied through UV-Vis, SEM,

FT-IR, and TGA techniques.

• Lamotrigine loading in Pluronic F127 is better than Pluronic

F68.

• Pluronic® polymer has good candidate for Drug delivery

applications for Lamotrigine.

Future possible study

• Cell viability and uptake study of Curcumin-loaded

Nanoparticles.

• In Vitro release study of Lamotrigine-loaded Nanoparticles.

• Proven track of nontoxicity of prepared Nanoparticles and

establishment of Pluronic® polymer has important candidate

for Drug delivery applications for both the drug.

curcumin

PEO PEOPPO

Micellisation

Core of micelle : PPO

Shell/corona of micelle : PEO

Curcumin Loaded nanoparticles

(CURNPs)

Lamotrigine Loaded nanoparticles

(LamoPNPs)

lamotrigine

Acknowledgments

I wish to acknowledge with thanks to Chemistry Department, kachcha university.

It is with deep sense of gratitude and profound appreciation toward my guide Dr.

Rakesh Sharma , that I present this Dissertation work. I feel highly indebted and

privileged for his valuable guidance, keen interest and constant encouragement

throughout my dissertation work.

I wish to acknowledge with thanks to Prof. P. T. DEOTA, Head Applied

Chemistry Department, for providing excellent laboratory facility and for

providing opportunity to dissertation work in my fourth semester.

I am also very thankful to research scholar Mr. Gautam Patel, Mr.Umesh

Chaudhari, and Mr. Deepak Singh for their guidance and support during my

dissertation work.

I am extremely indebted to research scholar Mr. Gautam Patel for carrying out

I.R & UV-Vis. analysis in due time.

…Thank you