Results & Discussion

Table of Contents
6. RESULTS AND DISCUSSION ........................................................................................................ 2
6.1. Preformulation Studies............................................................................................................... 2
6.1.1. Solubility studies .................................................................................................................. 2

6.1.2. Drug-excipients compatibility studies ................................................................................ 2

6.1.3. Development and validation of reverse phase HPLC method for estimation of
carboplatin...................................................................................................................................... 7

6.2. Formulation Development of Carboplatin PCL Nanoparticles............................................ 11

6.2.1. Formulation optimization of carboplatin-PCL nanoparticles by double emulsion
solvent evaporation technique .................................................................................................... 11

6.2.3. Evaluation of developed carboplatin PCL nanoparticles............................................... 19

6.3. Formulation Development of Carboplatin-PLGA 50:50 nanoparticles............................... 25

6.3.1. Formulation optimization of carboplatin-PLGA nanoparticles by double emulsion
solvent evaporation technique .................................................................................................... 25

6.3.2. Characterization of optimized carboplatin PLGA 50:50 nanoparticles ....................... 30

6.3.3. Evaluation of developed carboplatin-PLGA nanoparticles ........................................... 33

6.4. References.................................................................................................................................. 40

1
 Results & Discussion

6. RESULTS AND DISCUSSION

6.1. Preformulation Studies

6.1.1. Solubility studies

The solubility profile of carboplatin in water and at different pH conditions (ranging from pH
1.3 to 9.0) was thoroughly investigated. Table 6.1 shows the solubility profile of carboplatin
in water and different pH conditions. The carboplatin is found to be sparingly soluble in water
with a solubility of 17.681 mg/mL.

Table 6.1. Solubility of carboplatin in water and different pH conditions

Water/ Buffer (pH) Solubility (mg/mL)

[Mean±SDa]

Distilled Water 17.681 ±1.92

0.1 M HCL (1.3) 9.412±1.15

0.1M sodium acetate/ acetic acid (4.0) 8.273±0.92

0.1M Sodium carbonate/sodium hydrogen carbonate (9.0) 6.531±0.84

a
n=3, 24-h data.

The solubility of carboplatin in various buffers with pH ranging from 1.3 to 9.0 was between
5-10 mg/mL. The drug exhibited a solubility of 9.412 mg/mL, 8.273 mg/mLl and 6.531
mg/mL in pH 1.3, 4.0 and 9.0 respectively. The aqueous solubility of the drug may pose
serious encapsulation issues during formulation of carboplatin as polymeric nanoparticles.

6.1.2. Drug-excipients compatibility studies

The purpose of the drug-excipients compatibility studies is to check for any possible
interaction between carboplatin and other formulation components such as polymers,
stabilizer and cryoprotectant used in the development of carboplatin nanoparticles. The
mixtures of drug and individual excipients were prepared 1:1 ratio and were stored in stability
chamber at 25 0±2 0C/60 ± 5% RH for four week time period (Suriyaprakash et al., 2011).
After the study period the drug excipient mixtures were assed for the changes in physical
attributes as well as any chemical interactions using FTIR and DSC studies. The pure
carboplatin did not show any changes in the physical characteristics. Similarly carboplatin in

2
 Results & Discussion

the presence of other excipients in 1:1 ratio apparently did not show any characteristic change
in morphological characteristics. Therefore formulation excipients were found to be
compatible with drug substance.

6.1.2.1. Fourier Transform Infrared (FTIR) spectral studies

Compatibility of excipients with the pure drug was evaluated by analyzing the FTIR spectra
of physical mixtures (Figure 6.1.). FTIR spectrum of carboplatin exhibited an absorption band
at 3267.52 cm-1 which corresponds to N-H stretching frequency of amino groups. The
presence of carbonyl function is evident from characteristic absorption band at 1637.62 cm-1.
Characteristic peaks at 2991.69 and 2951.19 cm-1 corresponds to aliphatic C-H stretching
vibrations. IR spectrum of physical mixture of carboplatin and excipients in 1:1 proportions
showed all the characteristic peaks of the drug without any appreciable shift which indicates
that there is no significant interaction between the drug and the excipients. Physical mixture
of the drug with excipients such as PCL, PLGA, PVA, F-68 and mannitol in 1:1 ratios
exhibited peaks corresponding to the drug at 3280-3283 cm-1 (-N-H str), 1637-1643 cm-1
(>C=O str,) and 2910-2949 cm-1 which corresponds to aliphatic C-H stretching vibrations.

3
 Results & Discussion

Figure 6.1. FTIR spectra of pure carboplatin and physical mixture. Pure carboplatin (a),
Carboplatin+ PCL + PVA + Mannitol (CPCLP) (b), Carboplatin + PLGA 50:50 + PVA +
Mannitol (CPLGAP) (c), Carboplatin + PLGA 50:50 + F-68 + Mannitol (CPLGAF).

4
 Results & Discussion

6.1.2.2. Thermogram properties

DSC thermograms of free carboplatin (CRB) and excipients such as PCL polymer (PC), PLGA
50:50 (PLGA), PVA (PV) or pluronic F-68 and Mannitol (MN) were analysed to detect the
occurrence of interactions, if any, between the drug and excipients (Figure 6.2). DSC thermogram of
pure carboplatin exhibited endothermic thermal transitions at 259 0C which corresponds to the
melting temperature (Tm) of carboplatin (Parveen et al., 2010). The DSC thermogram of pure
polymer PCL showed an endothermic peak only at 65.5 0C which corresponds to its melting
temperature (Tm), while PLGA 50:50 showed glass transition temperature (Tg) at 50.5 0C.
Thermogram of PVA showed glass transition temperature (Tg) at 100 0C and the melting
endotherm was observed at 191.5 0C, while pluronic F-68 exhibited endothermic peak at 55.5 0C
which corresponds to its melting temperature (Tm). The DSC thermogram of mannitol exhibited an
endothermic peak at 171.5 0C which corresponds to its melting temperature (Tm).

Physical mixture of pure carboplatin along with these excipients did not show any significant
shift in peaks compared to that of pure drug and pure excipients. The thermogram of CPCLP
showed endothermic peaks at 66.02 0C, corresponds to melting endotherm (Tm) of PCL.
Another endothermic peak was observed at 172.02 0C which was the melting endothermic
peak of mannitol. A broad endothermic peak at 255.16 0C corresponds to slight preshifted
melting peak of carboplatin. Similarly other physical mixtures like CPLGAP (Figure 6.2c)
and CPLGAF (Figure 6.2d) also showed broad endothermic peaks at around 255 0C
corresponding to the melting endotherm of carboplatin. The results indicated no significant
interactions among the pure drug and excipients used in the formulation confirming the
compatibility between carboplatin and formulation components.

5
 Results & Discussion

Figure 6.2. DSC thermograms of pure carboplatin and physical mixture. Pure carboplatin (a),
Carboplatin+ PCL + PVA + Mannitol (CPCLP) (b), Carboplatin + PLGA 50:50 + PVA +
Mannitol (CPLGAP) (c), Carboplatin + PLGA 50:50 + F-68 + Mannitol (CPLGAF).

6
 Results & Discussion

6.1.3. Development and validation of reverse phase HPLC method for estimation of
carboplatin

6.1.3.1. Method development

Effect of change in pH of buffer

It was observed that when the pH of the buffer was 3.3, 3.5 and 3.7 carboplatin peak appeared
at 4.48 min, 4.24 min and 4.07 min respectively.

Effect of change in ratio of mobile phase

It was observed that when the composition of mobile phase was 85:15, 90:10 and 95:5 (%
v/v) the carboplatin eluted at 6.51 min, 4.24 min and 3.45 min respectively.

Effect of change in flow rate

It was observed that when the flow rate was varied from 0.9, 1.0 and 1.1 ml/min carboplatin
peak appeared at 5.82 min 4.24 min and 3.81 min respectively.

Effect of change in wave length

When the analysis was carried out at wavelength of 224, 229 and 234 nm carboplatin peak
appeared at 4.19, 4.24 and 4.33 nm respectively.
Based on the above optimization parameters, the following chromatographic condition was
selected for the estimation of carboplatin by HPLC method consists of:
Stationary phase : Hypersil C18 ODS column (250 x 4.6 mm, 5μm)
Mobile phase : Sulphuric acid 0 .001N and sodium sulphate 0.02M (pH 3.5) as
aqueous phase and methanol as the organic phase (90:10)
Run time : Isocratic run for 10 min
Detection wavelength : 229 nm
Flow rate : 1 mL/min
Injection volume : 20 μL
Temperature : Ambient (around 25 °C)

With the above separation condition, the retention time for carboplatin was found to be 4.2
min. The typical standard chromatogram of carboplatin is showed in the Figure 6.3.

7
 Results & Discussion

Figure 6.3. A representative chromatogram of carboplatin at the concentration of 32 µg/mL

6.1.3.2. Method validation

Linearity

The coefficient determination (r2) for the present method was 0.9998 which indicated that the present
method is linear and it is linear in the range from 0.5 – 250 μg/mL (y = 20774x – 10273). Acceptance
criteria for Linearity, (r2) is >0.999. Calibration curve is shown in the Figure 6.4.

Accuracy

The recoveries at three different levels (80, 100, 120 %) were found to be within the range of
98 to 102% as per ICH guidelines (Guideline, 2005). Mean % recovery (Mean±SD) was
found to be 99.43±0.81, shown in Table 6.2.

Table 6.2. The recoveries at three different levels

Accuracy level Amount added (mg) Amount recovered (mg) % Recovery

80%-1 4.01 3.99 99.75
80%-2 4.07 4.02 98.95
80%-3 4.05 4.03 99.56
100%-1 5.02 4.94 98.34
100%-2 5.05 5.04 99.72
100%-3 5.08 5.00 98.51
120%-1 6.08 6.03 99.15
120%-2 6.11 6.17 101.03
120%-3 6.04 6.03 99.83
Mean±SD 99.43±0.81

8
 Results & Discussion

Repeatability

The repeatability of the proposed method was determined from the percentage relative
standard deviation of six determinations at 100 % of test concentration. The % RSD was
found to be 0.607% which in within the acceptance criteria for the repeatability (<1% RSD).

Intermediate precision

The intermediate precision of the proposed method was determined from the overall
percentage relative standard deviation of six determinations at 100 % of test concentration on
different days by two different analyst. The overall % RSD was found to be 1.15 % which in
within the acceptance criteria for the intermediate precision (<2% RSD).

Specificity

There was no interaction observed between drug and excipients present in the formulation,
confirmed the specificity of the method.

Robustness

Robustness of the method was evaluated under following conditions:

Set 1: Normal conditions: Wavelength 229 nm; flow rate 1.0 ml/min, Mobile phase
composition 90:10, pH of buffer 3.5.
Set-2: Sample (pH of buffer 3.3)
Set-3: Sample (pH of buffer 3.7)
Set-4: Sample (Mobile phase 85:15)
Set-5: Sample (Mobile phase 90:5)
Set-6: Sample (flow rate 0.9 ml/min)
Set-7: Sample (flow rate 1.1 ml/min)
Set-8: Sample (detection wavelength 224 nm)
Set-9: Sample (detection wavelength 234 nm)

The % RSD and overall %RSD for each set is shown in Table 6.3.

9
 Results & Discussion

Table 6.3. The % RSD and overall %RSD for each set

Conditions % RSD Overall % RSD

Set-1 1.74 -
Set-2 1.41 1.45
Set-3 1.24 1.43
Set-4 1.37 1.09
Set-5 0.95 1.14
Set-6 0.70 1.20
Set-7 0.58 1.19
Set-8 0.54 1.24
Set-9 0.83 1.18

The overall percentage relative standard deviation in the various parameters was found to be
less than 2%. Thus the result indicated that the method was robust.

Table 6.4. Data of analytical method validation of carboplatin by HPLC

Validation Parameters Validation results Acceptance criteria

Linearity (r2) (0.05 – 250 μg/ml) 0.9998 > 0.999

Accuracy (% Recovery 99.43±0.81 98-102 %

Mean±SD)

Robustness (% RSD) < 2.0 < 2.0

Repeatability (% RSD) 0.607 < 1.0

Intermediate precision (% RSD) 1.15 < 2.0

LOD (μg/ml) 0.05 S/N ratio should be 3:1

LOQ (μg/ml) 0.2 S/N ratio should be 10:1

Limit of detection (LOD) and limit of quantitation (LOQ)

The present method LOD and LOQ was found to be 0.05 and 0.2 μg /ml respectively. The
percentage RSD for six injections of the LOQ solution was 1.59 %.

10
 Results & Discussion

6000000

5000000
Peak area 4000000

3000000

2000000

1000000

0
0 50 100 150 200 250 300
Concentration (mcg/ml)

Figure 6.4. Calibration plot of carboplatin by HPLC method

6.2. Formulation Development of Carboplatin PCL Nanoparticles

6.2.1. Formulation optimization of carboplatin-PCL nanoparticles by double emulsion
solvent evaporation technique

Various batches of carboplatin PCL nanoparticles were prepared by altering the experimental
conditions for the optimization of encapsulation efficiency and particle size of the
nanoparticles. Based on the results of solubility studies of drug and polymer in various
aqueous and organic solvents, carboplatin exhibited appreciable solubility in water and PCL
polymer was soluble in dichloromethane. The carboplatin loaded PCL nanoparticles prepared
using optimized parameters had a percentage entrapment efficiency of 27.95±4.21 % and
particle size of 311.6±4.7 nm, with a polydispersity index and zeta potential of 0.21±0.03 and
-16.3±3.7 mV. The particle size, PDI, zeta potential and % encapsulation efficiency results of
all batches are presented in Table 6.5.

Entrapment efficiency

To attain optimum entrapment of drug, various batches were prepared with different ratios of
drug: polymer and the ratio of 1:15 showed an optimum entrapment efficiency of 27.95±4.21%.
The entrapment efficiency of all designed batches of carboplatin PCL nanoparticles are reported
in Table 6.5. When the drug: polymer ratio was raised from 1:1 to 1:60, entrapment efficiency
increased but particle size also increased beyond drug: polymer ratio of 1:15. But further increase
in drug: polymer ratio showed a decrease in drug entrapment in nanoparticle and increase in

11
 Results & Discussion

particle size. This trend of increase in particle size with increase in polymer concentration is a well
reported phenomenon (Hoffart et al., 2002; Galindo-Rodriguez et al., 2004; Averineni et al.,
2012). During the encapsulation process the amount of drug loss can be reduced with increase in
polymer concentration to a particular limit.

Particle size, polydispersity index and zeta potential

Average particle size, polydispersity index and zeta potential of all batches of CPCs are
reported in Table 6.5. The polydispersity index (PDI) was measured to determine the range of
particle size distribution. Particle size distribution of optimized batch CPC-08 is depicted in
Figure 6.6. The PDI value for CPC-08 was found to be 0.21±0.03. Pattern of size distribution
of nanoparticles plays a vital role in defining the mucosal permeation, drug release behaviour
and their prospect for intranasal administration. Smaller sized particles are benefitted by EPR
effect as they are likely to accumulate in the tumour sites. Particles smaller than 1 μm can
pass through the nasal cavities along with the inspired air, whereas particles bigger than 10
μm are likely to be deposited at the anterior parts of the nose and thus evade ciliated
absorption areas (Illum et al., 1987). The zeta potential for optimized formulation CPC-08
(Figure 6.7) was -16.3±3.7 mV, which was due to the negative charge of PCL. Zeta potential
values in the range -15 to -30 mV are ideal for stabilized nanoparticles (Musumeci et al.,
2006). The electrostatic repulsion between particles with high negative zeta potential values
are more which prevents their aggregation and thereby stabilize the nanoparticulate dispersion
(Feng et al., 2001). The optimized formulations (CPC-07 & CPC-08) with desirable
properties were further used for in situ nasal perfusion studies.

Figure 6.6. Particle size distribution of carboplatin PCL nanoparticles (CPC-08)

12
 Results & Discussion

Figure 6.7. Zeta potential profile of carboplatin PCL nanoparticles (CPC-08)

Table 6.5. Formulation of carboplatin PCL nanoparticles

Average Zeta
Drug to Stabilizer/
Batch particle Potential Entrapment
Polymer Polymer Conc (% PDI *
Code size * *
efficiency(%) *
ratio w/v)
(nm) (mV)
CPC-01 PCL 1:1 PVA/1.0% 511.4±6.2 -11.6±3.4 0.22±0.034 11.12±2.11

CPC-02 PCL 1:5 PVA/1.0% 545.1±7.7 -14.3±3.1 0.29±0.079 12.98±2.70

CPC-03 PCL 1:10 PVA/1.0% 461.5±4.8 -14.7±5.1 0.21±0.009 14.25±3.63

CPC-04 PCL 1:15 PVA/1.0% 335.8±4.5 -15.6±2.4 0.26±0.017 15.69±3.13

CPC-05 PCL 1:30 PVA/1.0% 486.4±5.5 -12.7±3.7 0.29±0.056 16.23±4.24

CPC-06 PCL 1:60 PVA/1.0% 689.5±9.9 -11.2±1.3 0.27±0.064 17.15±4.60

CPC-07 PCL 1:15 PVA/1.5% 324.3±4.3 -15.5±4.1 0.299±0.03 22.5±5.14

CPC-08 PCL 1:15 PVA/2.0% 311.6±4.7 -16.3±3.7 0.21±0.03 27.95±4.21

CPC-09 PCL 1:15 PVA/2.5% 327.3±5.1 -13.7±2.4 0.24±0.05 25.68±6.48

CPC-10 PCL 1:15 F-68/1.0% 689.5±8.9 -14.7±1.9 0.34±0.082 11.87±4.93

CPC-11 PCL 1:15 F-68/1.5% 524.3±7.8 -14.6±3.1 0.29±0.071 13.12±5.45

CPC-12 PCL 1:15 F-68/2.0% 456.3±7.1 -15.8±1.8 0.28±0.065 19.43±4.47

*
These results are mean ± standard deviation. (n=3).

13
 Results & Discussion

Effect of drug: polymer ratio on encapsulation efficiency and particle size

The effect of drug : polymer ratio on entrapment efficiency and particle size was studied at
different drug : polymer ratios like 1:1, 1:5, 1:10, 1:15, 1:30 and 1:60. An increase in drug:
polymer ratio from 1:1 to 1:15 caused an increase in entrapment efficiency and decrease in
particle size. Nevertheless, with further escalation in the drug: polymer ratio, though the
encapsulation efficiency was found to improve, particle size also increased. (Budhian et al., 2007;
Shah et al., 2009). The reason for this may be that with rise in the polymer concentration, the
viscosity of the organic phase also increased leading to rapid diffusion of the organic phase into
the aqueous phase. Higher polymer concentrations resulted in the formation of coarse dispersions
and higher particle size, possibly due to the inadequate quantity of stabilizer present in the
aqueous phase. The results are shown in Figure 6.8 and 6.9.

800
Particle size (nm)

700
600
500
400
300
200
0 20 40 60 80
Polymer content (mg)/(mg) drug

Figure 6.8. Effect of drug: polymer ratio on particle size of carboplatin PCL nanoparticles
18

16
% EE

14

12

10
0 20 40 60 80
Polymer content (mg)/(mg) drug

Figure 6.9. Effect of drug: polymer ratio on entrapment efficiency of carboplatin

PCL nanoparticles

Effect of stabilizers on encapsulation efficiency and particle size

Stabilizers are used in formulation of nanoparticles so as to provide stability of the dispersion

in aqueous medium (Mainardes et al., 2005). Various surfactants like PVA and Pluronic F-68

14
 Results & Discussion

were tried to rule out the effect of type of surfactant on physicochemical properties of
carboplatin nanoparticles. PVA was selected as the stabilizer and as it gave lower particle size
and maximum encapsulation efficiency without particle aggregation. Various concentrations
of PVA ranging from 1.0 % w/v to 2.5% w/v were used in the formulation and its effect on
encapsulation efficiency and particle size were studied. The results are shown in Table 6.5
and Figures 6.10 & 6.11. The particle size decreased and entrapment efficiency increased
with increasing PVA concentration. This was true only up to 2% w/v of PVA (Dora et al.,
2010). An optimum concentration (2.0% w/v) of stabilizer led to a reduced size of NP,
whereas when concentrations below 2.0% w/v were used, the level of stabilizer was found to
be inadequate as it caused aggregation. This may be because the stabilizer was insufficient to
cover and stabilize the dispersed nanoparticle completely, causing them to aggregate and
exhibit larger size (Feng et al., 2001). Hence, 2.0% w/v PVA concentration was considered
optimum.

340
Particle size (nm)

335
330
325
320
315
310
0.9 1.4 1.9 2.4 2.9
Surfactant concentration (%w/v)

Figure 6.10. Effects of surfactant concentration on particle size of carboplatin PCL

nanoparticles
29

24
% EE

19

14
0.9 1.4 1.9 2.4 2.9
Surfactant concentration (% W/V)

Figure 6.11. Effects of surfactant concentration on entrapment efficiency of carboplatin

PCL nanoparticles

15
 Results & Discussion

Effect of homogenization speed and sonication amplitude

High speed homogenization technique was employed to obtain carboplatin PCL nanoparticles.
To optimize the homogenization conditions, homogenization was carried out at four different
speed levels (5000, 10000, 150000, 20000 rpm) by keeping other formulation parameters
constant. As the homogenization speed and sonication amplitude was increased, a decrease in
particle size was observed. However increase in homogenization speed above 20000 rpm and
sonication amplitude above 80 % does not show any decrease in particle size. Higher
homogenization speed can cause particle agglomeration due to high energy build up (Nafee et
al., 2007).

Effect of cryoprotectants during freeze drying process of carboplatin PCL nanoparticles

Various cryoprotectants such as sucrose, fructose, dextrose and mannitol with suitable
concentrations were tested for their effect on freeze drying process of carboplatin polymeric
nanoparticles. Among them mannitol yielded completely dry and free flowing powders with
lowest particle size. The concentration of mannitol was varied at different levels such as 2.5, 5
and 10 % w/v to study their influence on physicochemical properties of nanoparticles. 2.5 %
w/v mannitol produced a particle size of 751 nm with slight broad range of particles. However
lower particle size was observed with 5 and 10 % w/v mannitol displaying a particle size
distribution in the range of 310-515 nm. Therefore 5 % w/v mannitol was found to be
sufficient to obtain nanoparticles of desirable properties.

6.2.2. Characterization of optimized carboplatin PCL nanoparticles

6.2.2.1. Fourier Transform Infrared spectroscopic studies

FTIR spectrum of carboplatin loaded PCL nanoparticle (CPC-08) (Figure 6.12) was analysed
to prove the encapsulation of carboplatin in nanoparticles and to detect the occurrence of
interactions, if any, between the drug and excipients in the nanoparticles. The presence of
characteristic peaks of pure drug carboplatin in carboplatin loaded PCL nanoparticle (CPC-
08) can be confirmed by the typical bands at 3281.02 cm-1(-N-H str), 2949.26, 2910.68
cm-1(aliphatic -C-H str), 1725.35 cm-1(>C=O). Thus FTIR spectrum of carboplatin loaded
PCL nanoparticle (CPC-08) showed all the characteristic peaks of the drug without any

16
 Results & Discussion

appreciable shift which confirms the encapsulation of drug and also that there is no significant
interaction between the drug and the excipients during the formation of nanoparticles.

90

%T

75

661.61

578.66
773.48

632.67
2864.39
2910.68

727.19
933.58
1689.70
3402.54

1579.75

960.58

875.71
60
2949.26

1726.35

1637.62

1464.02
3281.02

1197.83

1041.60
1020.38
1247.99

1084.03
1288.49
45

1377.22
30
4000 3500 3000 2500 2000 1750 1500 1250 1000 750 500
08 1/cm

Figure 6.12. FTIR spectrum of CPC-08

6.2.2.2. Thermogram properties

DSC thermograms of PCL polymer (PC), free carboplatin (C), PVA (PV), mannitol (MN)
and carboplatin loaded PCL nanoparticles (CPC-08) were analyzed to study the existence of
interactions, if any, between the drug and excipients in the nanoparticles (Figure 6.13). DSC
thermogram of pure carboplatin exhibited endothermic thermal transitions at 259 0C which
corresponds to its melting temperature (Tm) (curve a). DSC thermogram of pure polymer
0
PCL only showed an endothermic peak 71-72 C which corresponds to its melting
temperature (Tm) (curve c). The thermogram of carboplatin loaded nanoparticles (CPC-08)
exhibited endotherm at 63.84 0C which corresponds to melting endotherms of PCL polymer
(curve b). The thermogram also showed a broad and very weak endotherm between 235-245
0
C corresponding to the melting endotherm of carboplatin. This may be due to the conversion
of drug from crystalline to amorphous form. Therefore it may be concluded that the
carboplatin in PCL nanoparticles existed in amorphous form of a molecular dispersion or
solid solution state in the polymer matrix (Jain et al., 2011). Thus it could lead to increased
solubility and finally to an improved biological activity (Dhanaraju et al., 2010).

17
 Results & Discussion

Figure 6.13. DSC thermogram of carboplatin (a), PCL polymer (C) and carboplatin loaded
PCL nanoparticlesCPC-08 (b).

6.2.2.3. Scanning electron microscopy (SEM)

The SEM photomicrograph of optimized carboplatin loaded PCL nanoparticles were obtained
before and after gold sputtering is shown in the Figure 6.14a & 6.14b. The SEM
photomicrograph of optimized carboplatin PCL nanoparticles confirmed their spherical shape
(Figure 6.14). The particles exhibited moderate uniformity and all the particles were discrete
entities without any aggregation. They were homogeneous with smooth surface and without
any rupture.

Figure 6.14a. Scanning electron microscopy of carboplatin loaded PCL nanoparticles (CPC-
08) after gold sputtering.

18
 Results & Discussion

Figure 6.14b. Scanning electron microscopy of carboplatin loaded PCL nanoparticles (CPC-
08) without gold sputtering.

6.2.3. Evaluation of developed carboplatin PCL nanoparticles

6.2.3.1. In vitro release studies

In vitro release study was performed for all batches prepared. CPC-07 and CPC-08 were found to be
the best batches and results are shown in Figure 6.15. In vitro release profiles for all the formulations
showed biphasic behaviour consisting of initial burst release followed by a sustained release phase
over a period of 72 h. The initial burst release of drug may be due to the release of carboplatin which
was loosely bound or adsorbed on the surface of the nanoparticles. Sustained release may be due to
slow diffusion of the drug from the highly lipophilic polymeric matrix. The release of entrapped
carboplatin from nanoparticles with lower concentration of polymer was more rapid than those with
higher polymer concentration. This could be due to the fact that increase in polymer concentration
leads to the formation of thick and less porous polymer matrix.

To study the drug release kinetics, the data obtained from in vitro drug release studies of
optimized batch CPC-08 was fitted to the different models like first-order and Higuchi’s
model. Regression coefficients (R2) for first-order and Higuchi’s model were found to be
0.9854 and 0.9984 respectively. Thus, in vitro drug release of CPC-08 formulation was best
explained by Higuchi kinetic diffusion control mechanism, as the plot showed the maximum
linearity (Jain et al., 2009).

19
 Results & Discussion

To study the mechanism of drug release, the data obtained from in vitro drug release studies
were fitted to the Korsmeyer–Peppas model. The regression coefficient for the plot of log
cumulative percentage drug release vs log time of the Korsmeyer–Peppas equation for
carboplatin PCL nanoparticle formulation CPC-08 was found to be 0.9981. The value of
release exponent (n) was found to be 0.5321. As the n value is >0.5, it can be concluded that
the release of carboplatin from PCL nanoparticle was by non-Fickian diffusion (diffusion
coupled with erosion) (Peppas, 1984; Vaghani et al., 2010).

Figure 6.15. In vitro % cumulative drug release profile of carboplatin PCL nanoparticle
formulations.

6.2.3.2. Ex vivo permeation studies of carboplatin PCL nanoparticles through sheep

nasal mucosa

The drug permeation pattern through sheep nasal mucosa reflected a similar release pattern as
that of in vitro release studies, but the amount of drug permeated was lower. This resistance
to the permeation of drug molecules could be attributed to the complexity of nasal mucosa in
terms of anatomy, chemical composition etc. (Tas et al., 2004). Between the two formulations
(CPC-07 & CPC-08) selected for the study, formulation CPC-08 exhibited better permeation
across the sheep nasal mucosa. The cumulative percentage of carboplatin permeated from
CPC-08 after 8 h was found to be 22.91±2.08%, upon three determinations. Permeation
profile is shown in Figure 6.16.

20
 Results & Discussion

Figure 6. 16. Percentage cumulative drug permeated from optimized batches of carboplatin
loaded PCL nanoparticles through nasal mucosa

Histopathological studies

Histopathological studies were carried out to examine the histological changes in nasal
mucosa caused by carboplatin PCL nanoparticles. The cross section of the sheep nasal
mucosa used for permeation before (untreated control, Figure 6.17a) and after the permeation
(treated) was stained by hematoxylin–eosin (HE) and observed under light microscope. Cross
section of control nasal mucosa showed ciliated respiratory epithelium and normal goblet
cells (Mahajan et al., 2012). The effect of carboplatin PCL nanoparticles (CPC-08) on sheep
nasal mucosa, 8h after permeation studies (treated, Figure 6.17b) showed no severe damage
on the integrity of nasal mucosa when compared to untreated control. A mild epithelial
disruption of nasal mucosa was observed.

Figure 6.17. Histopathology of sheep nasal mucosa. a) Nasal mucosa of untreated control
(20X) b) Nasal mucosa permeated with CPC-08 (20X).

21
 Results & Discussion

6.2.3.3. Nasal absorption studies of carboplatin PCL nanoparticles by in situ nasal

perfusion in Wistar rats

Nasal absorption of pure carboplatin solution and optimized carboplatin loaded nanoparticle
formulations (CPC-07 & 08) were studied by in situ nasal perfusion in Wistar rats. All the samples
showed progressive nasal absorption with time. A plot of percentage drug remaining in the
perfusing solution versus time is shown in Figure 6.18. Carboplatin loaded PCL nanoparticles
exhibited better nasal absorption compared to pure carboplatin solution. Between the two
formulations studied for nasal absorption, CPC-08 showed better nasal absorption profile compared
to CPC-07. The first order nasal absorption rate constants for carboplatin solution and optimized
formulations CPC-07 andCPC-08 were calculated from the slopes of first-order plots of the
percentage drug remaining in the perfusing solution versus time. The first order nasal absorption rate
constants for carboplatin solution, CPC-07 and CPC-08 were found to be 5.067 × 10-3, 6.218 ×10-3
and 6.909 × 10-3 min-1 respectively (Shinichiro et al., 1981; Huang et al., 1985). There is a
statistically significant (p < 0.05) increase in first order nasal absorption rate constant for optimized
carboplatin PCL nanoparticles (CPC-07 & CPC-08) when compared to that of pure drug solution.
Smaller particle size and bioadhesive nature of PCL polymer which enables it to bind to nasal
mucosa has increased the residence time and subsequently increased the nasal absorption (Dondeti et
al., 1996; Ugwoke et al., 2001).

Figure 6.18. Percentage of carboplatin remaining in the perfusate versus time after nasal
perfusion of rats with carboplatin, CPC-08 and CPC-07 (n=3).

6.2.3.4. In vitro cytotoxicity studies by MTT assay

For in vitro cytotoxicity study, cells were incubated with various concentrations of both pure drug
and CPCs for 48 h and 96 h separately. Percentage cell viability (Y-axis) was plotted against

22
 Results & Discussion

concentration of the carboplatin (X-axis). Concentration of carboplatin that reduces the viability
of cell to 50% (IC50 value) was determined for carboplatin and carboplatin nanoparticles after 48
h and 96 h treatment. Results reveal a dose dependent reduction in the percentage cell viability
after treatment with carboplatin nanoparticle or carboplatin (Figure 6.19). Pure carboplatin does
not show any significant difference in cytotoxicity after 48 h and 96 h of incubation. At shorter
incubation time (48 h) cytotoxicity of carboplatin nanoparticles CPC-08 & 07 were low at all
tested concentrations compared to that of carboplatin solution. The IC50 value for CPC-08 & 07
after 48 h incubation was found to be 10.80±1.03 and 11.974±1.15 μg/mL respectively, while for
pure carboplatin, the IC50 value was 6.738±0.74μg/mL. This may be due to the fact that polymeric
nanoparticles of carboplatin only release about 60 percent of drug from polymer matrix as
indicated by in vitro release studies on CPCs. For longer incubations (96 h) carboplatin
nanoparticle showed better cytotoxicity at all tested concentrations than that of pure carboplatin.
The result suggests that polymeric nanoparticles release the drug slowly and have increased
retention in the cells as compared to carboplatin alone (Averineni et al., 2012). Thus the IC50
value was significantly (p < 0.05) increased with carboplatin loaded nanoparticles compared to
pure carboplatin after 96 hours of incubation, indicating enhanced antitumor activity of
nanoparticles against human glioblastoma cells LN229 (Figure 6.20).

Figure 6.19. In vitro cytotoxicity studies of carboplatin nanoparticle in human glioblastoma

cells after 48h and 96h.

23
 Results & Discussion

Figure 6.20. In vitro cytotoxicity of pure carboplatin and carboplatin PCL nanoparticle formulations
in human glioblastoma cells (LN229).*P<0.05 versus carboplatin pure (after 96 h).

6.2.3.5. Accelerated stability studies

Stability studies of optimized batch of freeze-dried carboplatin nanoparticle CPC-08 was

carried out and stability of formulation was evaluated on the basis of particle size, zeta
potential, and entrapment efficiency as main parameters (Kalaria et al., 2009). Slight increase
in average particle size and a slight decrease in zeta potential and percentage entrapment
efficiency were observed on three-month storage. As there is no significant alteration in
average particle size, zeta potential and entrapment efficiency, it can be concluded that the
carboplatin nanoparticle formulation CPC-08 is stable at 25±2 0C / 60±5 % RH for a total
period of three months (Table 6.6).

Table 6.6. Accelerated stability studies of optimized batch of carboplatin PCL

nanoparticles

Stability parameter Test period

0 month 1 month 2 month 3 month

Particle size* (nm) 311.6±4.7 343.3 ± 11.2 385.2 ± 14.7 401.2 ± 10.5

Zeta Potential* (mV) -16.3±3.7 -15.7 ± 2.1 -14.5 ± 1.8 -13.3 ± 3.1

Entrapment efficiency (%)* 27.95±4.21 25.19 ± 2.76 24.27 ± 1.44 23.96 ± 2.93
*
These results are mean±standard deviation. (n=3).

24
 Results & Discussion

6.3. Formulation Development of Carboplatin-PLGA 50:50 nanoparticles

6.3.1. Formulation optimization of carboplatin-PLGA nanoparticles by double emulsion

solvent evaporation technique

In order to optimize the encapsulation efficiency and particle size of carboplatin loaded PLGA
nanoparticles; various batches were prepared with varying experimental conditions and were
evaluated. Based on the results of solubility studies of drug and polymer in various aqueous
and organic solvents, carboplatin was found to be soluble in water and PLGA 50:50 polymer
was soluble in dichloromethane. Based on the evaluation parameters such as particle size,
polydispersity index, zeta potential and entrapment efficiency, batch CAPG-6 was selected as
optimized batch of carboplatin nanoparticles using PLGA polymer. The carboplatin loaded
PLGA nanoparticles developed using optimized parameters had a percentage entrapment
efficiency of 31.93±2.59 % and particle size of 165.4±2.95 nm, with a polydispersity index
and zeta potential of 0.32±0.014 and -13.5±0.85 mV respectively. The particle size, PDI, zeta
potential and % encapsulation efficiency results of all batches are presented in Table 6.7.

Entrapment Efficiency

To attain optimum entrapment of drug, various batches were prepared with different ratios of
polymer to drug. Polymer to drug ratio of 1:15 showed an optimum entrapment efficiency of
31.93±2.59 %. The entrapment efficiency of all designed batches of carboplatin PCL
nanoparticles are reported in Table 1. As the drug: polymer ratio increased from 1:1 to 1:60,
entrapment efficiency increased but particle size also increased with increase drug: polymer
ratio. This trend of increase in particle size with increase in polymer concentration is a well
reported phenomenon (Hoffart et al., 2002; Galindo-Rodriguez et al., 2004; Averineni et al.,
2012). Therefore batch with lowest particle size and optimum entrapment efficiency was
selected as the optimised batch. The optimized formulation (CAPG-6) with highest
entrapment efficiency was further used for in situ nasal perfusion studies.

Particle size, polydispersity index and zeta potential

Average particle size, polydispersity index and zeta potential of all batches of carboplatin
PLGA 50:50 nanoparticles are reported in Table 6.7. The polydispersity index (PDI) was
measured to determine the range of particle size distribution. Particle size distribution and
zeta potential of optimized batch CAPG-6 is depicted in Figure 2&3. The PDI value for

25
 Results & Discussion

CAPG-6 was found to be 0.32±0.014. Pattern of size distribution of nanoparticles plays a vital
role in defining the mucosal permeation, drug release behaviour and their prospect for
intranasal administration. Smaller sized particles are capable of passing through the nasal
cavities with the inspired air, can move along the olfactory region and get distributed in the
brain and CSF and are benefitted by EPR effect as they tend to accumulate in the tumour sites
(Illum et al., 1987). The zeta potential for optimized formulation CAPG-6 was -13.5±0.85
mV, which was due to the negative charge of PLGA (Moreno-Vega et al., 2012). The
electrostatic repulsion between particles with high negative zeta potential values stabilize the
nanoparticulate dispersion and inhibits their aggregation (Feng et al., 2001).

Table 6.7. Formulation development of carboplatin PLGA 50:50 nanoparticles

Drug to Stabilizer/ Average Zeta Entrapment

Batch
Polymer Conc (% particle size * Potential * PDI *
efficiency
Code
ratio w/v) (nm) (mV) (%) *
CAPG-1 1:05 PVA/0.5% 190.9±2.34 -8.31±0.72 0.27±0.021 11.69±1.17

CAPG-2 1:10 PVA/0.5% 179.6±3.63 -9.85±0.81 0.19±0.013 13.71±1.29

CAPG-3 1:15 PVA/0.5% 180.8±2.71 -12.52±0.97 0.15±0.011 17.54±1.55

CAPG-4 1:30 PVA/0.5% 284.7±2.11 -13.31±1.11 0.29±0.023 19.81±1.67

CAPG-5 1:60 PVA/0.5% 419.7±3.77 -11.61±0.89 0.23±0.021 22.06±1.89

CAPG-6 1:15 PVA/2.0% 165.4±2.95 -13.5±0.85 0.32±0.014 31.93±2.59

CAPG-7 1:15 PVA/1.5% 205.3±2.95 -10.56±0.71 0.27±0.018 23.37±2.21

CAPG-8 1:15 PVA/1.0% 227.5±2.21 -11.54±0.92 0.31±0.027 22.76±1.99

CAPG-9 1:15 PVA/0.25% 301.5±2.95 -12.31±1.12 0.34±0.029 12.44±1.15

CAPG-10 1:15 F-68/2.0% 213.7±2.13 -15.61±1.31 0.37±0.032 25.73±2.37

CAPG-11 1:15 F-68/1.5% 291.7±2.85 -17.12±1.25 0.42±0.037 18.41±1.75

CAPG-12 1:15 F-68/1.0% 367.5±3.55 -19.9±1.35 0.48±0.039 15.16±1.41

CAPG-13 1:15 F-68/0.5% 443.4±4.27 -18.1±1.29 0.45±0.035 11.57±1.11

CAPG-14 1:15 F-68/0.25% 502.1±4.84 -19.5±1.37 0.41±0.031 9.21±1.05

*
These results are mean ± standard deviation. (n=3).

26
 Results & Discussion

Figure 6.21. Particle size distribution of carboplatin PLGA nanoparticles (CAPG-6)

Figure 6.22. Zeta potential profile of carboplatin PLGA nanoparticles (CAPG-6)

Effect of drug: polymer ratio on encapsulation efficiency and particle size

The effect of drug : polymer ratio on entrapment efficiency and particle size was studied at
different drug: polymer ratios like 1:5, 1:10, 1:15, 1:30 and 1:60. An increase in drug :
polymer ratio from 1:5 to 1:60 caused an increase in entrapment efficiency and increase in
particle size as reported earlier (Budhian et al., 2007; Shah et al., 2009). An increase in the
polymer concentration led to an increase in the viscosity of the organic phase, and this
resulted in rapid diffusion of the organic phase into the aqueous phase. Because of insufficient

27
 Results & Discussion

amount of stabilizer in aqueous phase for a given amount of polymer may cause an increase in
particle size at high polymer concentrations (Seju et al., 2011).

450
400
Particle size (nm) 350
300
250
200
150
100
0 10 20 30 40 50 60 70
Polymer content (mg)/(mg) drug

Figure 6.23. Effect of drug: polymer ratio on particle size of carboplatin PLGA nanoparticles

24
22
20
18
%EE

16
14
12
10
0 10 20 30 40 50 60 70
Polymer content (mg)/(mg) drug

Figure 6.24. Effect of drug: polymer ratio on entrapment efficiency of carboplatin PLGA
nanoparticles

Effect of stabilizers on encapsulation efficiency and particle size

Stabilizers are used in formulation of nanoparticles so as to provide stability of the dispersion

in aqueous medium (Mainardes et al., 2005). Various surfactants like PVA and Pluornic F-68
were tried to study the influence of type of surfactant on physicochemical properties of
carboplatin nanoparticles. PVA was selected as the stabilizer as it gave lower particle size and
maximum encapsulation efficiency without particle aggregation. It was tried at various
concentrations ranging from 0.25 % w/v to 2.0% w/v. The results are shown in Table 6.7 and

28
 Results & Discussion

Figures 6.25 & 6.26. The particle size decreased and Entrapment efficiency increases with
increasing PVA concentration. This was true only up to 2% w/v of PVA (Dora et al., 2010).
Further increase in PVA concentration does not show any appreciable effect on particle size
and encapsulation efficiency. An optimum concentration (2.0% w/v) of stabilizer led to a
reduced size of NP while the lesser amount of stabilizer yielded large sized particles. This
may be because the stabilizer was insufficient to cover and stabilize the dispersed nanoparticle
completely, causing them to aggregate and exhibit larger size. (Feng et al., 2001). Hence,
2.0% w/v PVA concentration was considered optimum.

350

300
Particle size (nm)

250

200

150

100
0 0.5 1 1.5 2 2.5
Surfactant concentration (%w/v)

Figure 6.25. Effect of surfactant concentration on particle size of carboplatin PLGA

nanoparticles

40
35
30
%EE

25
20
15
10
0 0.5 1 1.5 2 2.5
Surfactant concentration (% w/v)

Figure 6.26. Effect of surfactant concentration on entrapment efficiency of carboplatin PLGA

nanoparticles

29
 Results & Discussion

Effect of homogenization speed and sonication amplitude

High speed homogenization technique was employed to obtain carboplatin PLGA 50:50
nanoparticles. To optimize the homogenization conditions, homogenization was carried out at four
different speed levels (5000, 10000, 150000, 20000 rpm) by keeping other formulation parameters
constant. Decrease in particle size was observed with increase in homogenization speed and
sonication amplitude. However increase in homogenization speed above 20000 rpm and sonication
amplitude above 80 % did not show any decrease in particle size. Higher homogenization speed
can cause particle agglomeration due to high energy build up (Nafee et al., 2007).

Effect of cryoprotectants during freeze drying process of carboplatin PLGA

nanoparticles
Various cryoprotectants such as sucrose, fructose, dextrose and mannitol with suitable
concentrations were tested for their effect on freeze drying process of carboplatin polymeric
nanoparticles. Among them mannitol yielded completely dry and free flowing powders with
lowest particle size. The concentration of mannitol was varied at different levels such as 2.5, 5
and 10 % w/v to study their influence on physicochemical properties of nanoparticles. 2.5 %
w/v mannitol produced a particle size of 357 nm with slight broad range of particles. However
lower particle size was observed with 5 and 10 % w/v mannitol displaying a particle size
distribution in the range of 161-215 nm. Therefore 5 % w/v mannitol was found to be
sufficient to obtain nanoparticles of desirable properties.

6.3.2. Characterization of optimized carboplatin PLGA 50:50 nanoparticles

6.3.2.1. Fourier Transform Infrared spectroscopic studies

FTIR spectrum of carboplatin loaded PLGA 50:50 nanoparticles (CAPG-6) (Figure-?) was analysed
to prove the encapsulation of carboplatin in nanoparticles and to detect the occurrence of
interactions, if any, between the drug and excipients in the nanoparticles. The presence of
characteristic peaks of pure drug carboplatin (figure-?) in carboplatin loaded PLGA nanoparticle
(CAPG-6) can be confirmed by the typical bands at 3269.34 cm-1 (-N-H str), 2989.66, 2953.02 cm-
1
(aliphatic -C-H str), 1639.49 cm-1(>C=O) Thus FTIR spectrum of carboplatin loaded PLGA 50:50
nanoparticle (CAPG-6) showed all the characteristic peaks of the drug without any appreciable shift
which confirms the encapsulation of drug and also that there is no significant interaction between
the drug and the excipients during the formation of nanoparticles.

30
 Results & Discussion

Figure 6. 27. FTIR spectrum of CAPG-6

Thermogram properties

DSC thermograms of PLGA 50:50 polymer (PG), free carboplatin (C), PVA (PV), mannitol
(MN) and carboplatin loaded PLGA 50:50 nanoparticle (CAPG-6) were analyzed to study the
existence of interactions, if any, between the drug and excipients in the nanoparticles (Figure.
3). DSC thermogram of pure carboplatin exhibited endothermic thermal transitions at 259 0C
which corresponds to the melting temperature (Tm) of carboplatin. (curve a). DSC
thermogram of pure polymer PLGA only showed an endothermic peak 43.61 0C which
corresponds to glass transition temperature (Tg) (curve d). The DSC thermogram of mannitol
exhibited an endothermic peak at 171.50C which corresponds to its melting temperature (Tm)
(curve-b). The thermogram of carboplatin loaded PLGA nanoparticles (CAPG-6) (curve c)
exhibited endotherm at 43.840C which corresponds to glass transition temperature of PLGA
polymer. This thermogram did not show any detectable endotherm corresponding to the
melting temperature of free drug between 200–300˚C. This may be due to the conversion of
drug from crystalline to amorphous form. Therefore it may be concluded that the carboplatin
in PLGA nanoparticles existed in amorphous form of a molecular dispersion or solid solution
state in the polymer matrix (Jain et al., 2011). Thus it could lead to increased solubility and
finally to an improved biological activity (Dhanaraju et al., 2010).

31
 Results & Discussion

Figure 6.28. DSC thermogram of carboplatin (a), Mannitol (b) carboplatin loaded PLGA
nanoparticles CAPG-6 (c) and PLGA 50:50 polymer (d).

Scanning electron microscopy (SEM)

The SEM photomicrograph of optimized carboplatin loaded PLGA nanoparticles were

obtained before and after gold sputtering is shown in the Figure 6.29a & 6.29b. The SEM
photomicrograph of optimized carboplatin PLGA nanoparticles (CAPG-6) confirmed their
spherical shape. The particles exhibited moderate uniformity and all the particles were
discrete entities without any aggregation. They were homogeneous with smooth surface and
without any rupture.

Figure 6.29a. Scanning electron microscopy of carboplatin loaded PLGA nanoparticles

(CAPG-6) after gold spluttering.

32
 Results & Discussion

Figure 6.29b. Scanning electron microscopy of carboplatin loaded PLGA nanoparticles

(CAPG-6) without gold spluttering.

6.3.3. Evaluation of developed carboplatin-PLGA nanoparticles

6.3.3.1. In vitro release studies

In vitro release study was performed for all batches prepared. CAPG-6 and CAPG-7 were
found to be the best batches and results are shown in Figure 6.30. In vitro release profiles for
all the formulations showed biphasic behaviour consisting of initial fast release followed by a
sustained release phase over a period of 72 h. The initial fast release of drug may be due to the
release of carboplatin which was loosely bound or adsorbed on the surface of the
nanoparticles. Sustained release may be due to slow diffusion of the drug from the highly
lipophilic polymeric matrix. The release of entrapped carboplatin from nanoparticles with
lower concentration of polymer was more rapid than those with higher polymer concentration.
This could be due to the fact that increase in polymer concentration leads to the formation of
thick and less porous polymer matrix.

To study the drug release kinetics, the data obtained from in vitro drug release studies of
optimized batch CAPG-6 was fitted to the different models like, first-order and Higuchi’s
model. Regression coefficients (R2) for first-order and Higuchi’s model were found to be
0.9481 and 0.9769 respectively. Thus, in vitro drug release of CAPG-6 formulation was best

33
 Results & Discussion

explained by Higuchi kinetic diffusion control mechanism, as the plot showed the maximum
linearity (Jain et al., 2009).
To study the mechanism of drug release, the data obtained from in vitro drug release studies
were fitted to the Korsmeyer–Peppas model. The regression coefficient for the plot of log
cumulative percentage drug release vs log time of the Korsmeyer–Peppas equation for
carboplatin PLGA nanoparticle formulation CAPG-6 was found to be 0.9579. The value of
release exponent (n) was found to be 0.502. As the n value is >0.5, it can be concluded that
the release of carboplatin from PLGA 50:50 nanoparticle was by non-Fickian diffusion
(diffusion coupled with erosion) (Peppas, 1984; Vaghani et al., 2010).
90
80
% Cumulative drug release

70
60
CAPG-6
50
CAPG-7
40
30
20
10
0
0 10 20 30 40 50 60 70 80
Time (h)

Figure 6.30. In vitro % cumulative drug release profile of carboplatin PLGA 50:50
nanoparticle formulations.

6.3.3.2. Ex vivo permeation studies of carboplatin PLGA 50:50 nanoparticles through

sheep nasal mucosa

The drug permeation pattern through sheep nasal mucosa reflected a similar release pattern as
that of in vitro release studies, but the amount of drug permeated was lower. This resistance
to the permeation of drug molecules could be attributed to the complexity of nasal mucosa in
terms of anatomy, chemical composition etc. (Tas et al., 2004). Between the two
formulations (CAPG-6 & CAPG-7) selected for the study, formulation CAPG-6 exhibited
better permeation across the sheep nasal mucosa. The cumulative percentage of carboplatin
permeated from CAPG-6 after 8 h was found to be 29.12±1.90%, upon three determinations.
Permeation profile is shown in Figure 6.31.

34
 Results & Discussion

35
30

%Cumulative drug
25

permeated 20
15 CAPG-6
CAPG-7
10
5
0
0 1 2 3 4 5 6 7 8 9
Time (h)

Figure 6. 31. Percentage cumulative drug permeated from carboplatin PLGA nanoparticles
through nasal mucosa

Histopathological studies

Histopathological studies were carried out to examine the histological changes in nasal
mucosa caused by carboplatin PCL nanoparticles. The cross section of sheep nasal mucosa
used for permeation before (untreated control, Figure 6.32a) and after the permeation (treated)
was stained by hematoxylin–eosin (HE) and observed under light microscope. Cross section
of control nasal mucosa showed ciliated respiratory epithelium and normal goblet cells
(Mahajan et al., 2012). The effect of carboplatin PLGA nanoparticles (CAPG-6) on sheep
nasal mucosa, 8h after permeation studies (treated, Figure 6.32b) showed no severe damage
on the integrity of nasal mucosa when compared to untreated control. A slight epithelial
disruption of nasal mucosa was observed.

Figure 6.32. Histopathology of sheep nasal mucosa. a) Nasal mucosa of untreated control
(20X) b) Nasal mucosa permeated with CAPG-6 (20X).

35
 Results & Discussion

6.3.3.3. Nasal absorption studies of carboplatin PLGA nanoparticles by in situ nasal

perfusion in Wistar rats

Nasal absorption of pure carboplatin solution and optimized carboplatin loaded PLGA
nanoparticle formulations (CAPG-6 & 7) were studied by in situ nasal perfusion in male Wistar
rats. All the samples showed progressive nasal absorption with time. A plot of percentage drug
remaining in the perfusing solution versus time is shown in Figure 6.33. Carboplatin loaded
PLGA 50:50 nanoparticles exhibited better nasal absorption compared to pure carboplatin
solution. Between the two formulations studied for nasal absorption, CAPG-6 showed better nasal
absorption profile compared to CAPG-7. The first order nasal absorption rate constants for
carboplatin solution and optimized formulations CAPG-6 andCAPG-7 were calculated from the
slopes of first-order plots of the percentage drug remaining in the perfusing solution versus time
(Huang et al., 1985). The first order nasal absorption rate constants for carboplatin solution,
CAPG-6 and CAPG-7 were found to be 5.067 × 10-3, 9.212 × 10-3 and 7.83 × 10-3 min-1
respectively. There is a statistically significant (p < 0.05) increase in first order nasal absorption
rate constant for optimized carboplatin PLGA nanoparticles (CAPG-6 & CAPG-7) when
compared to that of pure drug solution. Smaller particle size and bioadhesive nature of PLGA
polymer which enables it to bind to nasal mucosa has increased the residence time and
subsequently increased the nasal absorption (Dondeti et al., 1996; Ugwoke et al., 2001).
100
CAPG-6
CAPG-7
90
Carboplatin
% Drug remaining

80

70

60

50

40
0 10 20 30 40 50 60 70 80
Time (min)

Figure 6.33. Percentage of carboplatin remaining in the perfusate versus time after nasal
perfusion of rats with carboplatin, CAPG-6 and CAPG-7 (n=3).
36
 Results & Discussion

A comparison of the nasal absorption of carboplatin from carboplatin loaded polymeric

nanoparticles (CPC-08 & CAPG-6) and that of pure carboplatin solution, it is observed that
CAPG-6 exhibited better nasal absorption (Figure 6.34). The first order nasal absorption rate
constant value for carboplatin loaded PLGA 50:50 nanoparticles (CAPG-6) was better than
that of carboplatin loaded PCL nanoparticles (CPC-08). This increased nasal absorption of
CAPG-6 may be due to comparatively faster release of carboplatin from PLGA 50:50
polymeric matrix than from PCL polymeric matrix. Pure carboplatin solution exhibited slower
nasal absorption, this may be due to the fact that absence of polymeric coating lowered the
residence time on nasal mucosa due to lack on bioadhesion.
100
CAPG-6

90 CPC-08
Pure carboplatin solution
% Drug Remaining

80

70

60

50

40
0 10 20 30 40 50 60 70 80
Time (min)

Figure 6.34. Percentage of carboplatin remaining in the perfusate versus time after nasal
perfusion of rats with pure carboplatin solution and carboplatin polymeric nanoparticles.

6.3.3.4. In vitro cytotoxicity studies by MTT assay

For in vitro cytotoxicity study, cells were incubated with various concentrations of both pure drug
and carboplatin loaded PLGA 50: 50 nanoparticles for 48 h and 96 h separately. Percentage cell
viability (Y-axis) was plotted against concentration of the carboplatin (X-axis). Concentration of
carboplatin that reduces the viability of cell to 50% (IC50 value) was determined for carboplatin
and carboplatin nanoparticles after 48 h and 96 h treatment. Results reveal a dose dependent
reduction in the percentage cell viability after treatment with carboplatin nanoparticle or

37
 Results & Discussion

carboplatin (Figure 6.35). Pure carboplatin does not show any significant difference in
cytotoxicity after 48 h and 96 h of incubation. At shorter incubation time (48 h) cytotoxicity of
carboplatin nanoparticles CAPG-6 & 7 were low at all tested concentrations compared to that of
carboplatin solution. The IC50 value for CAPG-6 & 7 after 48 h incubation was found to be
7.75±1.29 and 10.43±1.41 μg/ml respectively, while for pure carboplatin, the IC50 value was
6.738±0.74 and 6.188±0.602 μg/ml after 48 and 96 hrs of incubation respectively. This may be
due to the fact that polymeric nanoparticles of carboplatin only release about 70 percent of drug
from polymer matrix as indicated by in vitro release studies on carboplatin loaded PLGA 50: 50
nanoparticles. For longer incubations (96 h) carboplatin nanoparticle showed better cytotoxicity at
all tested concentrations than that of pure carboplatin (Averineni et al., 2012). The IC50 value for
CAPG-6 & 7 after 96 h incubation was found to be 3.54±0.55 and 4.48±0.49 μg/mL respectively.
The result suggests that polymeric nanoparticles release the drug slowly and have increased
retention in the cells as compared to carboplatin alone. Thus the IC50 value was significantly (p <
0.05) increased with carboplatin loaded nanoparticles compared to pure carboplatin after 96 hours
of incubation, indicating enhanced antitumor activity of nanoparticles against human glioblastoma
cells LN229 (Figure 6.36).

120

CAPG-6; 96 h
100
CAPG-6; 48 h
CAPG-7; 96 h
% Cell Viability

80 CAPG-7; 48 h
Carboplatin-pure; 48 h
60 Carboplatin-pure; 96 h

40

20

0
0 5 10 15 20 25
Conc μg/ ml

Figure 6.35. In vitro cytotoxicity studies of carboplatin nanoparticle in human glioblastoma

cells LN229.

38
 Results & Discussion

14
48 h
12 96 h

10
IC 50 (μg/ml)
8

4 *

0
Carboplatin-Pure CAPG-6 CAPG-7

Figure 6.36. In vitro cytotoxicity of pure carboplatin and carboplatin PLGA 50:50 nanoparticle
formulations in human glioblastoma cells (LN229).*P<0.05 versus carboplatin pure (after 96 h).

6.3.3.5. Accelerated stability studies

Stability studies of optimized batch of freeze-dried carboplatin PLGA nanoparticle CAPG-6

was carried out and stability of formulation was evaluated on the basis of particle size, zeta
potential, and entrapment efficiency as main parameters. Slight increase in average particle
size and a slight decrease in zeta potential and percentage entrapment efficiency were
observed on three-month storage. As there is no significant alteration in average particle size,
zeta potential and entrapment efficiency, it can be concluded that the carboplatin nanoparticle
formulation CPC-08 is stable at 25±2 0C / 60±5 % RH for a total period of three months
(Table 6.8).

Table 6.8. Accelerated stability studies of optimized batch of carboplatin PCL nanoparticles

Stability parameter Test period

0 month 1 month 2 month 3 month

Particle size* (nm) 165.4 ± 2.95 170.3 ± 4.11 174.4 ± 4.29 177.2 ± 5.01

Zeta Potential* (mV) -13.5±0.85 -12.9 ± 0.91 -11.8 ± 1.1 -11.3 ± 1.5

Entrapment efficiency (%)* 31.93±2.59 30.15 ± 2.48 29.61 ± 1.59 28.75 ± 1.62
*
These results are mean±standard deviation. (n=3).

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 Results & Discussion

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