PREPARATION AND CHARACTERIZATION OF CURCUMIN GOLD NANO SUSPENSION FOR BREAST CANCER

PREPARATION AND CHARACTERIZATION OF CURCUMIN GOLD

NANO SUSPENSION FOR BREAST CANCER

Pankti P. Dalwadi*1,2, Dr. Pragnesh Patani1 and Dr. Indrajeet Singhavi2

1,2

Department of Pharmacology, A-One Pharmacy College Naroda, Ahmedabad, Gujarat,

India.

2

Dean Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Pacific Academy of

Higher Education and Research, Udaipur, Rajasthan, India.

ABSTRACT

Breast cancer is world third leading cause of death in the world among

women. Breast cancer is spreads from milk duct. Nano suspension

improves bioavailability of poorly soluble drugs improves therapeutic

effectiveness of drug to target at the site of effectiveness. Curcumin

gold nano suspension (CGNS) is prepared by Bottom –Up technology.

Curcumin is obtained from rhizomes of curcuma longa. Curcumin

modulate anti cancer activity by activation of NF- κB pathway.Gold

has surface plasmon resonance property which reduces the dose of the

chemotherapy and radiation therapy. Curcumin gold nano suspension

solid particles were obtained by lyophilisation. Particle size of

optimized batch of CGNS is 322.6±4.98nm and poly dispersity index of 0.18±0.020. Zeta

potential of CGNS is -27.42 ±0.32. Differential scanning calorimetry shows that nano particle

was in amorphous in nature. Improves dissolution rate of CGNS because of reduces particle

size. Transmission Micrograph of the CGNS was show particle size of 322.6nm. The results

suggested that precipitation was an effective way to prepare CGNS improves the dissolution

rate for targeting cancer cells.

KEYWORDS: Curcumin, Gold nano particle, Precipitation method, Nano suspension,

Dissolution.

INTRODUCTION

Breast cancer is major health problem in developed and developing countries. As per

epidemiological survey various parameters such as mortality and survival rates varying

Article Received on 15 Nov. 2018,

Revised on 04 Dec. 2018, Accepted on 25 Dec. 2018

DOI: 10.20959/wjpr20191-13958

*Corresponding Author

Pankti P. Dalwadi

Department of

Pharmacology, A-One

Pharmacy College Naroda,

Ahmedabad, Gujarat, India.

between countries.[1] Breast cancer is accredited to various factors such as life style changes dietary changes, obesity, hormonal imbalance as well as exposure to radiation, alteration

menstrual changes. A Women with predispose to genetic mutation such as BRCA 1 and

BRCA 2. Current therapy of breast cancer is based on complementary and alternative

medicine .treatment approach for breast cancer.[2] It compiles both and involves different therapies such as surgery, Radiation and oncological treatment such as chemotherapy,

hormonal therapy and immunological therapy or systemically destroy or control cancer cell

throughout the body. Gold is newer approach for the treatment of cancer. Gold particles are

smaller in size and penetrate throughout the body preferentially accumulates at the tumor site

owing to EPR effect and importantly they can bind to many proteins and actively targeted to

cancer cells and by over expressing cell surface receptor.[3] Gold has characteristics of surface plasmon resonance and as chemically inert for the proteins, antibodies and genes and also has

higher biologically compatible and provide high surface area so large amount of drug can be

loaded.[4] Natural phytoconstitute from various medicinal plant has now widely interested as having promising effect in development of new class of anticancer drugs since they have

multiple targets in cancer cells with minimum toxicity of the constitute. curcumin is obtained

from rhizomes of turmeric curcuma longa which is widely used as Indian spice traditional

medicine for various disorders.[5] curcumin has anti antioxidant effect. Curcumin modulates biological pathways involved in mutagenesis, cell cycle regulation, apoptosis, angiogenesis,

tumour genesis and metastasis.[6] curcumin with paclitexal in combination produce increases antitumor efficacy. Curcumin reduces tumor growth and cell proliferation as well as

suppression of angiogenesis by intra peritoneal administration of curcumin in xenograft

model of breast cancer.[8] Reduces human epidermal receptors HER2 expression as well as phosphorylation of Akt MAPK in different cell line. curcumin induce apoptosis by inhibiting

fatty acid synthase.[9]

A nano suspension is a submicron colloidal dispersion of drug particle. It is a very finely

colloid, biphasic, dispersed and solid drug particle in aqueous vehicle, size below 1 µm,

stabilized by surfactants and polymers.[10] The particle size distribution of the solid particle in nano suspension is usually less than one micron with an average particle size ranging

between 200 to 600nm.[11] Nano suspension approach improves bioavailability of drug. Nano suspension approach is most suitable for the drug with high log P value and high melting

Nano suspension is prepared by various approaches like Top-down technology, bottom-up

technology, micro emulsion and emulsion as template.[13] In the present research work nano suspension was prepared by bottom -up technology in which drug is dissolved in organic

solvent which is then added to non organic solvent that cause precipitation of the nano

particle and the system is stabilized by stabilizer or surfactant to prevent further

agglomeration of particle.[14] The anti solvent approach precipitation approach is cost effective and rapid method.[15]

MATERIAL AND METHOD

Material

Gold chloride was procured from Astron Chemicals, Ahmedabad. Curcumin was procured

Yucca Enterprise, Mumbai, India. Poloxamer-188 was procured from Astron Research

Center. Ahmedabad, Gujarat. PVP K-90 was procured from SD chemicals, Mumbai.

Hydroxy propyl methyl cellulose was procured as gift sample from Cadila pharma, Gujarat

Indi.

Method

Preparation of Gold nano particle (GNP):

All glassware’s were cleaned with an aqua regia solution (1:3 nitric acid /hydrochloric acid).

1mM solution of 250 ml of hydrogen tetrachloroaurate trihydrate was brought to a boil for

vigorous stirring. To this solution 25ml of 33.8 mM of sodium citrate was added. Sodium

citrate is used as reducing agent. Gold solution was yellow at starting of reaction. Solution is

allow to boil. After adding sodium citrate act as reducing agent which sodium citrate turned

to citric acid.[16] Conversion of Au+3 to Au0 yellow coloured solution become transparent and colorless and It changed to black than slowly to wine red. The reaction can be summarized as.

2HAuCl4 + 3C6H8O7 (citric acid) 2Au + 3C5H6O5 + 8HCl+3CO2

Nano suspension was prepared by “Bottom –Up” technology. In the first step of Curcumin

was dissolved in the acetone to prepare organic phase and solution was then filter to clear

impurities at room temperature.[17] Different concentration of surfactant were dissolve in water as anti solvent and to this equal quantities of gold nano particle was added in different

batches. In second step drug was dispersed in gold nano particle using overhead stirrer at

2000 RPM for 24 hr leading to precipitation of nano suspension of phytoconstituents. The

Formulation

Ingredients F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16

Drug

(mg/ml) 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30

Poloxamer

-407(g/ml) 0.25 0.5 1 5 - - - -

Poloxamer-188(g/ml) - - - - 0.25 0.5 1 5 - - - -

PVP-K-90

(g/ml) - - - 0.25 0.5 1 5 - - - -

HPMC(g/ml) - - - 0.25 0.5 1 5

Acetone (ml) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

GNP (ml) 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

Water (ml) 05 20 25 50 05 20 25 50 05 20 25 50 05 20 25 50

CHARACTERIZATION OF PARTICLE SIZE

Particle Size and Polydispersity Index

Mean particle size of nano suspension and poly dispensability index of formulations were

determined by Malvern Zetasizer using water as dispersion medium. The sample of different

batches were scanned for determination of particle size. The samples of batches were

measured after appropriate dilution with distilled water. Mean particle size and distribution of

particle size in nanodispersion measured by dynamic light scattering. The zeta sizer measure

particle in the range of 0.6 nm- 6µm. The reading of samples were taken at 900 angle of incident beam. The zeta potential was measured by laser doppler anemometer coupled with

the same instrument. For accuracy the analysis of the sample was carried out in triplicate.[18]

Zeta potential

Zeta potential of the suspension is measured by Malvern zeta sizer. The zeta sizer is mainly

consist of the laser which is used to provide light source to illuminate particle within the

sample. For measurement of zeta potential light splits into an incident and reference beam.

The incident beam passes through the center of the sample cell. Zeta sizer produces a

frequency spectrum from which the electrophoretic mobility hence zeta potential is

calculated. The mobility and potential of nano dispersion particles with in range of 3nm-10

µm.[19]

Total Drug Content

An aliquot (0.5 ml) was evaporated to dryness. The residue was dissolved in acetone and

filtered with 0.45µm filter paper. The sample was analyzed using UV spectrophotometer at

Total Drug Content= (Total volume of nano suspension * Amount of drug in aliquot

)/Volume of aliquot.[20]

Entrapment Efficiency

High concentration of the free drug present in the supernant after centriguation can

determined by entrapment efficiency.10 ml of the freshly prepared nano suspension was

centrifuged at 10,000 rpm for 10 minutes using micro centrifuge. The supernant was

collected and unincorporated drug measured by taking absorbance of supernatant at 411 nm

by using UV spectro photo metrically.

Fourier Transform Infrared Spectroscopy

The Fourier transform infrared spectroscopy (FT-IR) spectra were scanned in the range of

3600-400cm-1 at an ambient temperature. FT-IR was scanned in the sequence of Curcumin, Polymer, Gold chloride and optimized batch of formulation using KBr technique. The pellet

were prepared using KBr hydraulic press under hydraulic pressure of 150 kg/m2.

Differential Scanning Calorimetry

DSC of pure Curcumin, Polymer, Gold chloride and Curcumin gold nano suspension were

studied using DSC (Shimadzu 60 with TDA trend line software, Shimadzu Co. Kyoto,

Japan). In DSC analysis, the samples were weighed (5mg) hermetically sealed in flat bottom

aluminum pans were heated over a temperature range of 50 ℃ to 300 ℃ at a constant

increasing rate of temperature of 10℃/min. Nitrogen gas 50 mL/min. Calibration of

instrument with respect to temperature and enthalpy was achieved.[21]

Transmission Electron Microscopy

Transmission electron microscopy (TEM) studies were performed using transmission

electron microscope(Philips Technai-20). The liquid nano suspension formulation was

dropped on copper-gold carbon grid and allowed to dry. This grid was then mounted in the

instrument and photographs were taken at various magnifications.[22]

In Vitro Drug Release Study

Dissolution studies were carried out in dissolution apparatus (TDT-08L, Electro lab) by using

the USP apparatus 2 paddle method. The bath temperature and paddle speed were set at 37℃

and 50 rpm respectively. The dissolution media include 900 ml of distilled water and

during dissolution process. An accurately weight bulk drug and nano suspension were

dispersed to the vessels. 5 ml samples were withdrawn at 5,10,20,30 40, 50, 60 min from

dissolution medium and replace with same buffer solution. The accumulation of dissolution

amount of Curcumin was determined by Spectro photo metrically.[24, 25]

RESULT AND DISCUSSION

Curcumin gold nano suspension was prepared by Bottom –Up approach. Polymers, solvent

and anti solvent ratio were selected based on preliminary formulation study of CGNS

batches. Nano suspension can be visualized by bluish white transparent ring appearance at the

top. Characterization of particle size of different batches are as seen in table 2:

Table 2: Particle Size, Poly dispersity index and Zeta potential of different batches.

BATCH NO PARTICLE SIZE(nm) (MEAN±S.D) PDI (MEAN±S.D) ZETA POTENTIAL(mV) (MEAN±S.D)

F1 608.5±5.88 0.46±0.01 -9.42±0.17

F2 570.9±12.71 0.35±0.02 -10.59±0.27

F3 322.6±4.98 0.18±0.02 -27.42 ±0.32

F4 472.4±5.19 0.25±0.03 -20.42±0.21

F5 695.3±10.27 0.53±0.04 -4.38±1.92

F6 600.1±11.72 0.43±0.03 -12.27±0.17

F7 547.8±3.68 0.35±0.03 -20.53±0.22

F8 215.7±6.9 0.36±0.05 -22.54±0.14

F9 425.9 ±3.29 0.27±0.05 -4.79±1.52

F10 393.7±6.54 0.71±0.03 -15.64±0.16

F11 372.3±10.78 0.57±0.06 -22.49±0.22

F12 188.6±3.26 0.45±0.02 -20.37±0.15

F13 1107.4±4.18 0.68±0.03 -12.65±0.39

F14 906.9±3.68 0.48±0.04 -12.81±0.20

F15 847.3±5.71 0.37±0.04 -19.52±0.19

F16 650.1±4.98 0.53±0.04 -23.81±0.15

Determination of Particle Size

Particle size of different batches were taken are as below. Batch size from F1 to F16,

maximum particle size was observed of batch F13 because of low concentration of polymers

induce aggregation of particles. The optimized shows particle size 322.6 nm as shown below

Polydispersity Index

Poly dispersity index of nano suspension batches were found to be in the range of o.71 to

0.18. F3 batch show the particle size distribution 0.18 which is most suitable for even

distribution of particle size.

Zeta Potential

Zeta potential means the distribution of charges over dispersed particle of nano suspension. It

indicates stability of nano suspension. Zeta potential of formulation batch of F1 to F16 were

found to be -9.42 to -27.42. F3 batch of formulation show zeta potential of -27.42 was found

to be stable curcumin gold nano suspension.

Figure 1: Particle size graph of optimized batch of curcumin gold nano suspension.

Total Drug Content

Drug content of all CGNS formulation was found to be greater than 80% except some of

batches indicates suitability of these method for particle size reduction. The optimized batch

Graph 1: Percentage total drug content of different CGNS batches.

Entrapment Efficiency

Entrapment efficiency all the batches were found to be greater than 60% except some of

batches. The optimized batch F3 shows entrapment efficiency 89.15% as shown in graph2.

Graph 2: Percentage entrapment efficiency of different CGNS batches.

Fourier Transform Infrared Spectroscopy

FTIR Spectrum of curcumin, polymer and nano suspension were taken to determine

physicochemical property and intermolecular bonding of drug and polymer. The FTIR

spectra of pure curcumin and curcumin gold nano suspension curves were well matched.

FTIR spectra shows O-H stretching was shift from 3299 cm-1 to 3205 cm-1 indicates intermolecular bonding. Broadening of peak was due to loading of curcumin with gold and

Figure 2: FT-IR spectra of pure Curcumin.

Figure 3: FT-IR spectra of Poloxamer 188.

Figure 5: FT-IR spectra of Curcumin gold nano suspension.

Differential Scanning Calorimetry

DSC performed to determine molecular state of the drug and to elucidate the physical nature

of drug in formulation. The DSC curve of Curcumin, Poloxamer 188, Gold nano particle and

Curcumin gold nano suspension (CGNS) and gold nano particle were carried out. Curcumin

exhibit endothermic peak at its melting point at 180.66 ℃ indicates the crystalline nature of

the drug. curcumin gold nano suspension showed melting peak at 175.14 ℃ indicates the

absence of the crystalline nature of the drug. Reduction in height of the peak this changes

indicates as result of reduction of particle size. curcumin loaded gold nanoparticle

endothermic peak at 115.18 ℃. This indicates changes in nature of drug giving a more

amorphous type as the product this may increase rate of dissolution of CGNS.(Fig:6).

50.00 100.00 150.00 200.00 250.00 300.00 Temp [C ]

-10.00 0.00 10.00 20.00 mW DSC

81.33x100_C

180.66x100_C

Thermal Analysis Result

50.00 100.00 150.00 200.00 250.00 300.00

Temp [C ] -40.00

-30.00 -20.00 -10.00 0.00 mW DSC

124.99x100_C

Thermal Analysis Result

FIGURE 6: DSC Thermogram of A) Poloxamer -188 B) Curcumin C) Gold Nano

Particle (D).

Transmission electron microscopy

TEM image of curcumin gold nano suspension indicate that curcumin particle exhibit

spherical shape with a diameter of about 322.6 nm. It shows uniform dispersion of particles.

The results suggested that curcumin nano particles were well stabilized by precipitation

method.(Fig:7).

B

Figure 7: TEM micrograph of optimized formulation batch.

In Vitro Drug Release

Most important feature of nano particle is the increase in dissolution of formulation with

decreasing in particle size. As decrease in particle size increase surface area for absorption

because of increase saturation solubility. In vitro drug release from the nano suspension were

carried out for 60 min. It was graphically represented as percentage drug release versus time

in minutes. The percentage of drug release was carried out of pure drug, physical mixture

formulation batch of CGNS. From study reveals that F3 batch shows faster release of drug as

compare to pure drug and physical mixture. Optimized formulation batch was enhanced

dissolution by 85% in 60min as compared to pure Curcumin showed 65%. By reduction of

particle size of formulation faster the action at target site of cancer in 60 min. As shown in

Graph 3.

CONCLUSION

Curcumin gold nano suspension was precipitated by solvent –anti solvent precipitation

method. Gold loaded curcumin target the cancer cells. The mean particle size of amporphous

CGNS was about 322.6nm with a uniform dispersion of particles.Smaller particle size and

large surface area of nano particle enhances dissolution rate of CGNS. DSC therm of the

CGNS indicates amorphous nature of the nanoparticle. This study demostrated the usefulness

of the precipitation technique to enhance dissolution of drugs.

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