Development and characterization of Centchroman

loaded PLGA Nanoparticles

1. Introduction

1.1 Nanoparticles

Nanotechnology is a rapidly expanding area, encompassing the development of man-made

materials in the 5–200 nanometer size range. This dimension vastly exceeds that of standard

organic molecules, but its lower range approaches that of many proteins and biological

macromolecules

Products of nanotechnology are expected to revolutionize modern medicine, as evidenced by

recent scientific advances and global initiatives to support nanotechnology and nanomedicine

research. The field of drug delivery is a direct beneficiary of these advancements. Due to their

versatility in targeting tissues, accessing deep molecular targets, and controlling drug release,

nanoparticles are helping address challenges to face the delivery of modern, as well as

conventional drugs. Since the majority of drug products employ solids, nanoparticles are

expected to have a broad impact on drug product development. In pharmaceutics, 90% of all

medicines, the active ingredient are in the form of solid particles. With the development in

nanotechnology, it is now possible to produce drug nanoparticles that can be utilized in a variety

of innovative ways.

Numerous investigations have shown that both tissue and cell distribution profiles of different

drugs can be controlled by their entrapment in submicrone colloidal systems (nanoparticles). The

rationale behind this approach is to increase efficacy, while reducing systemic side-effects.

Nanoparticulate drug delivery systems have been studied for several decades now, and many of

the features that make them attractive drug carriers are well known.

1.2 Advantages of nanoparticles in drug delivery

Large surface-to-volume ratio resulting enhanced interaction sites

Surface fictionalization for targeting

High payload and controlled release of drugs.

More efficient uptake by cells

1.3 Types of Nanoparticles

Liposomes

Nano-powders

Micelle

Polymeric nanoparticles

Dendrimers

Fullerenes

Metal nanoparticles

Magnetic nanoparticles

Biological nanoparticles

1.4 PLGA nanoparticles in cancer therapy

Polymeric nanoparticles provide significant flexibility in design because different polymers from

synthetic or natural sources can be used. Polymeric nanoparticles may represent the most

effective nanocarriers for targeted drug delivery. Some common polymers used for nanoparticle

formation include polylactide-co-glycolide (PLGA), polylactic acid, dextran, and chitosan.

Biodegradable polymers are typically degraded into oligomers and individual monomers, which

are metabolized and removed from the body via normal pathways.

Degradation and drug release kinetics can be precisely controlled by the physicochemical

properties of the polymer, such as molecular weight, polydispersity index, hydrophobicity, and

crystallinity. In general, drugs can be released in a controlled manner following Fickian kinetics

due to drug diffusion through the polymeric matrix, or be triggered in response to environmental

stimuli or released in the course of chemical degradation. The nanoparticle surface may be

sterically stabilized by grafting, conjugating, or adsorbing hydrophilic polymers, such as

polyethylene glycol (PEG), to its surface, which can reduce hepatic uptake and improve the

circulation half-life of the nanoparticles. PLGA is one of the most commonly used FDA

approved biodegradable and biocompatible polymers.

Nanoparticle-based drug delivery systems have many advantages for anticancer drug delivery,

including an ability to pass through the smallest capillary vessels, because of their very small

volume, and being able to avoid rapid clearance by phagocytes, so that their presence in the

blood stream is greatly prolonged. Nanoparticles can also penetrate cells and gaps in tissue to

arrive at target organs, including the liver, spleen, lung, spinal cord, and lymph. They may have

controlled-release properties due to their biodegradability, pH, ions, and/or temperature

sensitivity. All these properties can improve the utility of anticancer drugs and reduce their toxic

side effects.

1.5 Surface modification of PLGA nanoparticles

PLGA nanoparticles linked to targeting ligands are used to target malignant tumors with high

affinity. PLGA nanoparticles also have large surface areas and functional groups for conjugating

to multiple diagnostic (e.g., optical, radioisotopic, or magnetic) agents. Nanoparticle carriers

have high stability in biological fluids, and are more able to avoid enzymatic metabolism than

other colloidal carriers, such as liposomes or lipid vesicles.

Surface charges of nanoparticles also have an important influence on their interaction with the

cells and on their uptake. Positively charged nanoparticles seem to allow higher extent of

internalization, apparently as a result of the ionic interactions established between positively

charged particles and negatively charged cell membranes. Moreover, positively charged

nanoparticles seem to be able to escape from lysosomes after being internalized and exhibit

perinuclear localization, whereas the negatively and neutrally charged nanoparticles prefer to co-

localize with lysosomes. PLGA nanoparticles have negative charges which can be shifted to

neutral or positive charges by surface modification, for example PEGylation of the PLGA

polymer or chitosan coating respectively.

2. Centchroman

2.1 General Information & Properties

CAS Name: rel-1-[2-[4-[(3R,4R)-3,4-Dihydro-7-methoxy-2,2-dimethyl-3-phenyl-2H-1-

benzopyran-4-yl]phenoxy]ethyl]pyrrolidine.

Additional Names: (trans)-1-[2-[p-(7-methoxy-2,2-dimethyl-3-phenyl-4-

chromanyl)phenoxy]ethyl]pyrrolidine; 3,4-trans-2,2-dimethyl-3-phenyl-4-[p- -

pyrrolidinoethoxy)phenyl]-7-methoxychroman; trans-centchroman; ormeloxifene.

Trademarks: Centron (Torrent); Saheli (Hindustan Latex).

Molecular Formula: C30H35(NO)3.

Molecular Weight: 457.60

Derivative Type: Hydrochloride

CAS Registry Number: 51023-56-4

Manufacturers' Codes: 6720 CDRI

Molecular Formula: C30H35(NO)3.HCl

Percent Composition: C 72.93%, H 7.34%, N 2.84%, O 9.72%, Cl 7.18%

Properties:

White crystals; melting point 165-166°; UV max (methanol): 205, 280 nm; pKa 2.1; Soluble in

10 parts chloroform, 20 parts acetone, 60 parts 95% ethanol, 20 parts methanol; Practically

insoluble in water, isobutanol, 0.1N HCl, 0.1N NaOH; LD50 i.p. in mice: 400 mg/kg.

Activity and Mechanism: Contraceptives, Disorders of Sexual Function and Reproduction,

Treatment of, ENDOCRINE DRUGS, ONCOLYTIC DRUGS, Antiestrogens

Centchroman (CC) [C30H35O3N·HCl; trans-1-[2-{4-(7-methoxy-2,2-dimethyl-3-phenyl-3,4-

dihydro-2H-1-benzopyran-4-yl)-phenoxy}-ethyl]-pyrrolidine hydrochloride, 67/20; INN:

Ormeloxifene] is a non-steroidal antiestrogen and extensively used as a female contraceptive in

India.

There are reports of prevention of breast cancer by Centchroman. It has shown regression of

breast cancer lesion as well as anti mutagenic properties in bacterial mutagenicity assay and

mutation assays in female mice (Giri AK, Mukhopadhya A, Sun J, Hsie AW, and Ray S.1999).

In one of the study Centchroman has shown anti-neoplastic activity similar to Tamoxifen

irrespective of estrogen receptor status in breast cancer cell lines (Nigam M, Ranjan V,

Srivastava S, Sharma R, Balapure AK.2008). The mechanism cited is the caspase dependent

apoptosis. Centchroman has also shown its worth in treatment of mastalgia and fibroadenoma of

breast. In a study group of 60 patients Centchroman was proved as a safe drug in comparison to

Danazol and Bromocriptine which are the currently used drugs for the above conditions (Dhar A,

Srivastava A.2007)

3. PLGA Properties

3.1 Chemical structure

*

CH3

O

O

O

O

*

YX

Figure 1 Chemical structure of Poly (D, L-lactide-co-glycolide) (PLGA)

3.2 Chemical formula [C3H4O2]x[C2H2O2]y

3.3 Properties

Ratio of lactide: glycolide is 50:50

Molecular weight (Mw): 7000-17, 000

Transition temperature (Tg) : 42-46 oC

State of form: amorphous, light yellow in colour

Viscosity : 0.16-0.24 dL/g, 0.1 % (w/v) in chloroform (25oC)

4. Objective of Current Study

The objectives of the present study are:

a. Development and optimization of nanoparticles of centchroman

b. Characterization of particles with respect to size, zeta potential, drug loading and

entrapment efficiency.

c. Performing in-vitro dissolution studies and comparing the release rate with respect to pure

drug.

5. Experimental section

5.1 Materials

Pure reference standard of Centchroman (purity >99%) was provided by department of medicinal

and process chemistry of CDRI. PLGA (50:50), Tween 80, PVA and PEI were purchased from

sigma (USA). The water used in all experiments was prepared in a three-stage Millipore Milli-Q

plus 185 purification system (Bedford, US) and had a resistivity greater than 18.2 mW/cm.A

0.22µm cellulose membrane (Whatman International Ltd., Mailstone, England) was used for

filtration of buffer. Parafilm (Parafilm “M” Laboratory Film, American Can Company, CT, and

USA) was used for sealing tubes. All the solvents used were HPLC grade.

5.2 HPLC instrumentation and chromatographic conditions

5.2.1 Equipment:

Centchroman concentrations in the samples were measured by reverse-phase HPLC. The HPLC system

was equipped with 10 ATVP binary gradient pumps (Shimadzu), a rheodyne (Cotati, CA, USA) model

7125 injector with a 20 ml loop and SPD-M10 AVP UV detector (Shimadzu). HPLC separation was

Achieved on a Lichrosphere Lichrocart C18 column (250mm, 4mm, 5mm) (Merck). Column effluent

was monitored at 225 nm. Data was acquired and processed using Shimadzu (LC solution) software.

5.2.2 Preparation of mobile phase:

Mobile phase consist of acetonitrile: triple distilled water (80:20) (800µl TMAH added in 1 liter buffer

solution). The solution was degassed by using sonicator at 10% amp for 10 min before use.

Chromatography was performed at 250C at a flow rate of 1.5 ml/min on RP C18 column.

5.2.3 Preparation of stock solution

5mg of Centchroman was dissolved in 1ml of acetonitrile to give the concentration of 5mg/ml of stock

solution. Further, a working stock was prepared of 1μg/ml from which working standard was prepared

in the range of 200-1000 ng/ml. Twenty micro liters of the sample was injected in to the column.

Chromatographic conditions used in the analysis are given bellow.

5.2.4 Chromatographic conditions

Sr. No. Condition Description

1 Column specifications

MERCK 50225, Purospher

LichroCART RP18(15 cm×4.6 mm )

2 Pressure 100-200 kgf/cm2

3 Temperature 25OC

4 Flow Isocratic

5 Detector UV Visible Detector;

6 Mobile phase Acetonitrile: triple distilled water (80:20).

(800ul TMAH added in 1 liter buffer

solution)

7 Flow rate 1.5 ml/min.

8 Wavelength 205 and 280nm

5.3 Preparation of centchroman nanoparticles

Nanoparticles were prepared by using two different methods:

5.3.1 Emulsification-solvent evaporation (by sonication)

Sonication involves preparation of an organic phase consisting of polymer (PLGA) and drug

(Centchroman, typical concentration, 0.5 mg/ml) dissolved in DCM (typical volume1 ml). This organic

phase was added to an aqueous phase containing PEI as stabilizer to form an emulsion.

This emulsion was broken down into nanodroplets by applying external energy (through a homogenizer

or a sonicator) and these nano-droplets form nanoparticles upon evaporation of the highly volatile

organic solvent. The solvent was evaporated while magnetic stirring at 300 rpm under atmospheric

conditions for 4 hours leaving behind a colloidal suspension of PLGA nanoparticles in water.

5.3.2 Nano-precipitation

Nano-precipitation is similar to sonication, except that the organic solvent is acetone, a water miscible

solvent, and there is no application of external energy. Acetone was removed using rotavapor by

keeping the formulation overnight at room temperature to remove the traces. Once the colloidal

suspension of nanoparticles are prepared using one of the above three methods, the free drug is

removed by using our extraction method (Budhian et al., 2005) to obtain the final nanoparticulate

suspension containing encapsulated drug.

5.4 Particle Size Analysis

The developed nanoparticles were subjected to particle size analysis at different homogenization

conditions using Malvern Zetasizer Nano-Zs. (Malvern Instruments Inc., UK) for their size and

size distribution. Before performing analysis samples were diluted (20 times) with TDW. Size

and measurements were carried out using 1.52 refractive index of material as well as 1.32 RI for

dispersant (TDW) at 0.01 % absorbance. Count rate for sample was found about 200-220 at

attenuator position 7. Samples were measured thrice and average particle size was expressed as

the mean diameter of formulated nanosuspension.

5.5 Polydispersity index (PDI)

PDI was calculated at the same time as particle size using the same Zetasizer Nano-Zs (Malvern

Instruments Inc., UK.) Each sample was measured three times and an average PDI expressed as the

mean diameter.

5.6 Zeta potential (ZP)

The Zeta Potential is a measure of the electric charge at the surface of the nanoparticles indicating

physical stability of colloidal systems. The ZP values were assessed by determining the particle

electrophoretic mobility in respective of applied potential on the positive and negative electrodes.

Optical properties of the sample were defined on the basis of refractive index. Three observations were

recorded for each sample.

6. Determination of drug entrapment and drug loading

Entrapment efficiency was determined by centrifugation using both direct and indirect technique.

Direct Method:

In direct method drug-loaded NPs were separated from supernatant using centrifugation at

25000rpm for 45 minutes and the obtained pellet was dissolved in acetonitrile and analyzed by

reversed-phase (RP) HPLC system using Shimadzu HPLC system with LC software coupled to

UV detector.

Indirect Method:

In the indirect method, concentration in supernatant was determined. The drug entrapment and

drug loading was calculated by formula mentioned in equation below. All separations were

achieved on the Lichrosphere Lichrocart C18 column (250mm, 4mm, 5mm) (Merck) maintained

at 25°C. Acetonitrile: Triple Distilled Water (80:20) was used as mobile phase at flow rate of 1.5

ml/ min. The detection wavelength was 205 nm.

7. In-vitro release study

Dissolution studies were performed on 8 paddle dissolution apparatus (Labindia, India, USP

Type II, Disso 2000) at a rotation speed of 100 rpm. The dissolution was performed using 500 ml

of Phosphate buffer pH 7.4 containing 1% tween80 as a dissolution medium for 8 hours. All

dissolution tests were performed in triplicate containing centchroman in nanoparticles as well as

plain drug. At each predetermined sampling time, 1 mL of sample was withdrawn using

sampling port and the same volume was replaced to keep the total volume constant. Samples

were centrifuged, and 20 μl of resulting supernatant was injected into the HPLC for analysis.

8. Stability studies

Accelerated stability studies of nanoparticles were carried out following the protocols reported in

literature over a period of 1 and half months. Nanoparticles were transferred to 5 ml glass vials

sealed with plastic caps and were kept in stability chamber with temperature of 4±20C and

25±2°C. The formulations were monitored for changes in particle size, PDI and entrapment

efficiency. The physical appearances, ease of reconstitution were also recorded.

9. Results & Discussions

9.1. HPLC method development

Using pre-described HPLC conditions Centchroman was separated on the C-18 column. The

retention time was 15-16 min.

Table 9.1: Observed peak area with respect to concentration observed

9.2. Calibration curve of Centchroman

Calibration curve was plotted between the mean peak area at 205 nm with their respective

concentrations (ng/ml). Measurements were performed in triplicate.

Concentration (ng/ml) Area of peak (n=3) SD % SD

200 34407 ±1088.36 3.16

400 70415 ±1962.34 2.78

600 97619 ±3264.97 3.39

800 126906 ±5149.87 4.19

1000 158605 ±4997.5 3.15

Fig 9.1: HPLC Calibration curve of Centchroman.

Chromatogram: An HPLC chromatogram of Centchroman in Methanol, using Methanol

as blank

Fig 9.2: HPLC Chromatogram of Centchroman.

y = 152.44x + 6125R² = 0.9982

0

20000

40000

60000

80000

100000

120000

140000

160000

180000

0 200 400 600 800 1000 1200

Are

a

concentration(ng/ml)

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Volts

-0.0010

-0.0005

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

0.0035

Volts

-0.0010

-0.0005

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

0.0035

2.2

67

3943

6.1

3

14.2

83

60340

93.8

7

Detector A - 1 (280nm)

CENHTCHROMAN

10 ug

Retention Time

Area

Area Percent

9.3 Formulation optimization

Formulation optimization involves the effect of different parameters like effect of preparation

method, stabilizer concentration, and drug to polymer ration on particle size, zeta potential, PDI

and drug loading to get the best results.

9.3.1 Effect of preparation method:

Particle size affects the biopharmaceutical, physicochemical and drug release properties of the

nanoparticles. It is an important parameter because it has a direct relevance to the stability of the

formulation. Larger particles tend to aggregate to a greater extent compared to smaller particles,

thereby resulting in sedimentation. In the method, amount of stabilizer used and the ratio of drug

to polymer also affect the properties of nanoparticles. Particle size distribution and zeta potential

reveals the physical stability of the formulation i.e. surface charge of the particles control the

physical stability. Zeta potential also determines the behavior of the system on in-vivo

administration.

The choice of particular method for encapsulation of drug substance in a colloidal carrier is most

commonly determined by the solubility characteristics of the drug and polymer. The influence of

the preparation method on the particle size for a given composition of 1.25 mg drug, 1mg/ml PEI

and 25 mg polymer on Particle size and PDI is illustrated in Table 9.2. Nanoparticles prepared

with the nano-precipitation method were of smaller size than with the simple emulsion

technique. The method of preparation was also much simpler than emulsion technique. Hence,

the nanoprecipitation method was chosen for the further optimization of PLGA nanoparticles.

But with both techniques, nanoparticles exhibited a narrow size distribution (polydispersity index

0.2). Nanoparticles obtained by both techniques exhibited zeta potential in between 50-55 mV.

Table 9.2: Effect of method on size and PDI

Nano-precipitation Emulsification

Particle size (nm) 180±6.2 280±10.4

PDI 0.191±0.07 0.224±0.14

9.3.2 Effect of stabilizer:

Stabilizers in formulations have significant effect on stability of formulations. If the

concentration of stabilizer is too low, aggregation of the polymer will take place, whereas, if too

much stabilizer is used, drug incorporation could be reduced as a result of the interaction

between the drug and stabilizer. PEI was used as stabilizer because of its cationic nature and

highly efficient stabilizing properties. But as the concentration of stabilizer increased from

0.25mg/ml to 1mg/ml entrapment efficiency and particle size decreases significantly.

Table 9.3: Effect of PEI concentration

PEI concentration Particle Size

(nm)

PDI Zeta potential

0.25 mg/ml 271±9.4 0.432±0.22 + 55.5 ± 5.1

0.50mg/ml 234±6.8 0.361±0.12 + 58.4 ± 8.6

1.00mg/ml 180±6.01 0.283±0.09 + 62.8 ±9.5

PEI-Polyethyleneimine

9.3.3 Effect of various polymer to drug ratio:

It was observed that as drug: polymer (Centchroman: PLGA) ratio increased with constant

concentration of stabilizer from 0.04 to 0.3 particle size increased significantly and drug loading

also increased but thereafter, further increase in drug : polymer ratio showed reduced or

insignificant change in the drug entrapment efficiency. Drug loading increased from 2.8 to 20.4

% w/w and particle size increased from 180nm to 300nm.

Table 9.4: Effect of drug to polymer ratio on particle size and drug loading

Drug: polymer Particle size(nm) Drug loading w/w%

0.04:1 180±3.2 2.8

0.12:1 210±3.9 7.2

0.2:1 250±10.1 12.8

0.3:1 310±9.2 20.4

Fig 9.3: Size measured using Malvern Zetasizer Nano-Zs. (Malvern Instruments Inc., UK)

Fig 9.4: Zeta Potential measured using Malvern Zetasizer Nano-Zs.(Malvern Instruments Inc., UK)

Fig 9.5: Size obtained by nanoprecipitation method using acetone (in red) compared to that by

emulsification method by DCM (in green).

9.4 In vitro release profile

The drug released was studied as a function of time. Nanoparticles containing the minimum (4

%) and maximum PRZ loading (28%) (Theoretical loadings) were studied. The results over 32h

are shown in Fig. 9.3. The results of the assay show that there was a pronounced time

prolongation of drug release from nanoparticles in relation to the non-encapsulated drug. While

about 100% of non-encapsulated drug were found after approximately 6 h, only 31± % and

44±% of centchroman were released from nanoparticles after 24 h from batches containing,

respectively, 4 and 28 % of drug. The release pattern was much more sustained from

nanoparticles.

Fig 9.3: In vitro release kinetics from nanoparticles.(F1: 4 % theorical loading, F2: 28 % theoretical

loading)

9.5 Stability studies

The stability of the formulation analyzed by measuring their size, zeta potential and drug

entrapment at different storage conditions are presented in Fig 9.4a and Fig 9.4b. There was

practically no change in particle size, zeta potential and PDI after storage for 6 weeks at 4 ± 2°C.

0

20

40

60

80

100

120

0 10 20 30 40

% d

rug

rele

ase

d

Time (hrs)

F2

F1

Plain drug

However when these nanoparticles stored at 25± 2 OC for 6 weeks there was only slight increase

in both particle size as well as PDI. This stability believed to occur by their zeta potential of +

60±5 as zeta potential > 20 mV imparts long term stability for colloids.

Fig 9.4a: Stability studies at 40C

Fig 9.4b: Stability studies at 250C

0

0.05

0.1

0.15

0.2

0.25

100

120

140

160

180

200

220

240

0 1 2 4 6

PD

I

Size

(n

m)

Number of weeks

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

100

120

140

160

180

200

220

240

0 1 2 4 6

PD

I

Size

(n

m)

Number of weeks

Conclusion:

Centchroman is practically water insoluble drug, thus development of its PLGA nanoparticles

represents an effective formulation approach for in-vivo delivery. The different factors that affect

the physicochemical characterization of nanoparticles were the method of preparation used,

stabilizer concentration as well as drug to polymer ratio.

a. It was found that particles obtained with nano-precipitation were smaller in size as

compared to the emulsification method.

b. By increasing the concentration of PEI particle size decrease but entrapment efficiency

also decrease because of the more solubilizing effect of PEI on centchroman.

c. By increasing the drug to polymer ration, loading increase but parallel size of the

particles also increased.

d. Stability studies of 6 weeks revealed that nanoparticles were highly stable which may be

because of the high zeta potential.

e. In vitro release profile revealed that drug release was sustained for 32 hours whereas

plain drug dissolved completely in 5 hours.

References

1.Banker G S; Pharmaceutical applications of controlled release – an overview of the past,

present and future. In Medical Applications of Controlled Release; Langer, R. S. Wise, D. L.

Eds; CRC Press: Boca Raton, Florida, 1984; 11, Chapter 1.

2.Surendiran, S. Sandhiya, S.C. Pradhan& C. Adithan, Novel applications of nanotechnology in

medicine, Indian J Med Res. 2009; (130), 689-701.

3. Margaret A. Phillips a, Martin L. Granb, Nicholas A. Peppas, Targeted nanodelivery of drugs

and diagnostics, Nano Today. 2010; 5: 143-159.

4. Mauro Ferrari, Cancer Nanotechnology: Opportunities and challenges, Nature reviews. 2005;

5: 161-171

5. J. Braunecker, M. Baba, G.E. Milroy, R.E. Cameron The effects of molecular weight and

porosity on the degradation and drug release from polyglycolide Int. J. Pharm., 282 (2004), pp.

19–34

6. V.P. Torchilin, Drug targeting, Eur. J. Pharm. Sci. 11 (Suppl. 2) (2000) S81–S91. [31] J.

Davda, V. Labhasetwar, Characterization of nanoparticle uptake by endothelial cells, Int. J.

Pharm. 233 (1–2) (2002) 51–59.

7. S.K. Sahoo, J. Panyam, S. Prabha, V. Labhasetwar, Residual polyvinyl alcohol associated with

poly(D,L-lactide-co-glycolide) nanoparticles affects their physical properties and cellular uptake,

J. Control. Release 82 (1) (2002) 105–114.