1. 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

2.  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.

3. 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.

4. 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.

5. 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 o
C
 State of form: amorphous, light yellow in colour
 Viscosity : 0.16-0.24 dL/g, 0.1 % (w/v) in chloroform (25o
C)

6. 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.

7. 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 250
C 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 25O
C
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

8. 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.

9. 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.

10. 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±20
C 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.

11. 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

12. 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 + 6125
R² = 0.9982
0
20000
40000
60000
80000
100000
120000
140000
160000
180000
0 200 400 600 800 1000 1200
Area
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
2.2673943
6.13
14.28360340
93.87
Detector A - 1 (280nm)
CENHTCHROMAN
10 ug
Retention Time
Area
Area Percent

13. 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.

14. 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.

15. 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)

16. 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).

17. 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
%drugreleased
Time (hrs)
F2
F1
Plain drug

18. However when these nanoparticles stored at 25± 2 O
C 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 40
C
Fig 9.4b: Stability studies at 250
C
0
0.05
0.1
0.15
0.2
0.25
100
120
140
160
180
200
220
240
0 1 2 4 6
PDI
Size(nm)
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
PDI
Size(nm)
Number of weeks

19. 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.

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