CHAPTER - VI RESULTS AND DISCUSSIONshodhganga.inflibnet.ac.in/bitstream/10603/5358/15/15_chapter...

Chapter 6 Results & Discussion

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Enhanced Antitumour Effect of Camptothecin Loaded in Long Circulating Polymeric Micelles

CHAPTER - VI

RESULTS AND DISCUSSION

6.1 STANDARD GRAPH OF CAMPTOTHECIN

The λmax of camptothecin was found to be 371nm (The Merck Index.,

2001 ) which was selected for further analysis. Linearity range was obtained in

the concentration range between 5-35 µg/ml. Results were detailed in table 6 and

standard graph was given in Fig 8.

Table 6: Standard Graph of Camptothecin with PBS (pH 7.4)

S. No Concentration (µg/ml) Absorbance at 371 nm

1 0 0

2 5 0.173

3 10 0.316

4 15 0.489

5 20 0.578

6 25 0.709

7 30 0.849

8 35 0.954


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Fig 8: Standard Plot of Camptothecin

Straight line equation : y = 0.134x + 0.0168

Correlation coefficient : r2 = 0.994

6.2 OPTIMIZATION OF FORMULATION VARIABLES IN CAMPTOTHECIN

POLYMERIC MICELLES

CPT as a model drug was loaded into PMs by emulsification solvent

evaporation (ESE) method at various CPT: polymer ratios. The CPT loaded Me-

PEG-b-PCL micelles were characterized by measuring encapsulation efficiency,

in vitro drug release and in vivo studies. The ESE method followed has several

advantages over other methods including easier scale-up and less chance for

drug loss during encapsulation (Hamidreza Montazeri alibadi et al., 2006).

In order to achieve uniform particle shape and size and to improve drug

loading and encapsulation efficiency, various CPT-PMs formulation parameters


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were optimized in the study by trial and error method. PMs were prepared using

two different molecular weight di block copolymers (Me PEG5000-b-PCL13000 and

Me PEG5000-b-PCL5000) to investigate the influence of length of diblock

copolymers in drug loading.

Chloroform : acetonitrile or THF, were used as the organic co-solvents to

dissolve the block copolymers. The selection of organic solvent was based on its

miscibility with water, ability to dissolve both CPT and Me-PEG-b-PCL and a low

boiling point, to facilitate its evaporation and complete removal (Hamidreza

Montazeri Alibadi et al., 2006).

Two different organic solvents were selected to dissolve the block

copolymers, in order to investigate whether organic solvents have influence over

the formation of polymeric micelles.

6.2.1 Rotational speed of Rotary Flash Evaporator (RFE)

In order to evaporate the solvents more quickly and ensure stability of the

formulation, the RFE was attached to a vacuum pump; the temperature in water

bath and the rotation of the flask was optimized. Various rotation speeds (75 rpm,

100 rpm and 150 rpm) and temperature (40ºC and 70ºC) were tested. At higher

temperature fusion of micelles occurred and their shapes were not discrete. The

temperature of 70ºC used in hydration process is above the melting temperature

of Me PEG-b-PCL. When the formulation was heated at 70 ºC under high speed,

the mixture of Me PEG-b-PCL and CPT could interact with each other

adequately. When the temperature was lowered below their melting point, the Me

PEG-b-PCL began to load the drug in its rigid core when PEG still remained in a

liquid state in aqueous environment (Leyang Zhang et al., 2004).

At the temperature of 40º C and rotational speed of 100 rpm the polymeric

micelles were found to be stable with only a small amount of aggregated micelles.


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6.2.2 Sonication Time in Bath Sonicator

Sonication was done using a bath-type sonicator at various times

(2, 5 and 10 minutes). At 10 min and 5 min, the PMs were found to be unstable

and at the end of sonication there were no micelles seen. When the sonication

time was reduced to 2 min, however, the micelles were stable and of discrete

shape, had little particle aggregation and the size had also been reduced. Hence

the sonication time was fixed at 2 min.

6.2.3 Ultra Centrifugation to Separate the Unentrapped Drug

Refrigerated centrifugation was selected in the study to separate the PMs

from free CPT and other particles. Initially, the preparations were centrifuged at

6000 and 10,000 rpm at 4ºC for half an hour and there was no separation of PMs.

At 13,200 rpm at 4ºC for 1 hour, however, the PMs separated effectively from

free CPT. The PMs settled were collected and stored for the other studies.

For our final optimized procedure to prepare PMs with block copolymers,

we used two different molecular weight copolymers (Me PEG5000 -b-PCL13,000 and

Me PEG5000 -b-PCL5000 ), a sonication time of 2 min, a temperature in the RFE of

40ºC at 100 rpm and centrifugation at 13,200 rpm for 1 hr at 4ºC. Hydration

medium used was PBS pH 7.4. These parameters were kept constant for all the

formulations for reproducibility.

6.3 VESICLE SIZE, POLYDISPERSITY INDEX AND ZETA POTENTIAL

Surface morphology was determined by scanning electron microscopy

and vesicle size was determined by optical microscopy and transmission electron

microscopy specified in sections 5.7, 5.9 and 5.10.

Particle size and polydispersity index of prepared polymeric micelles

containing CPT were determined as per the procedure specified in section 5.8.


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Table 7: Particle Size and Polydispersity Index of Polymeric Micelles

Containing Camptothecin

Formulation code Mean particle diameter (µm) Polydispersity index

F1 0.437 ± 0.10 0.31 ± 0.02

F2 0.408 ± 0.09 0.34 ± 0.04

F3 0.369 ± 0.17 0.41 ± 0.09

F4 0.197 ± 0.16 0.29 ± 0.03

F5 0.332 ± 0.05 0.44 ± 0.08

F6 0.296 ± 0.11 0.46 ± 0.06

F7 0.241 ± 0.13 0.52 ± 0.07

F8 0.178 ± 0.09 0.47 ± 0.06

Table 8: Particle Size and Polydispersity Index of Polymeric Micelles

Containing Camptothecin

Formulation code Mean particle diameter (µm) Polydispersity index

F9 0.921 ± 0.15 0.86 ± 0.11

F10 0.853 ± 0.21 0.75 ± 0.03

F11 0.743 ± 0.23 0.71 ± 0.12

F12 0.799 ± 0.19 0.85 ± 0.09

F13 0.648 ± 0.25 0.70 ± 0.05

F14 0.615 ± 0.11 0.66 ± 0.13

Data Inference from Table 7 & 8

Since particle size has a crucial impact on the in vivo fate of a particulate

drug delivery system, control over the particle size is of great importance for all

drug carriers (Leyang Zhang et al., 2004). The results showed that PMs prepared


78


with THF showed higher average diameter with higher polydispersity when

compared with PMs containing chloroform: acetonitrile. It can be seen that the

higher the copolymer concentration in organic phase, the smaller the polymeric

micelle (PI-F4, F8, PII-F11, F14, 1:25).

However, at very low concentrations of block copolymer (drug: polymer

ratio 1:2.5), there was no polymeric micelles formed and hence polymeric micelle

containing THF was prepared further with the drug: polymer ratios of 1:5, 1:12.5

and 1:25.

The CPT PMs containing F4 (Me PEG 5000-b-PCL 13,000, 1:25 ratio) and F8

(Me PEG 5000 -b- PCL 5000, 1:25 ratio) showed smaller particle size and narrow

size distribution when compared with other formulations. From the zeta potential

measurement, it could be found that the zeta potential of micelles containing

F4 (PI, 1:25) and F8 (PII, 1:25) was in the range of -6.8 and -7.05 respectively.

The increase of zeta potential might be related to the shielding of ion charge by

PEG shell because of its non-ionic nature (Leyang Zhang et al., 2007).

The size of the polymeric micelles gradually increased with increasing

molecular weight of the hydrophobic segments of the Me PEG-b-PCL block

copolymers. However, in the preparation of CPT PMs using THF as a solvent, a

large amount of aggregation was found when compared with PMs containing

chloroform: acetonitrile.


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Photomicrographs of Camptothecin Polymeric Micelles Containing Tetra

Hydro Furan

Fig 9: F9, (1:5)

Fig 10: F10, (1:12.5)

Fig 11: F11, (1:25)

Fig 12: F12, (1:5)

Fig 13: F13, (1:12.5)

Fig 14: F14, (1:25)


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Photomicrographs showed that the CPT-PMs prepared using tetrahydro

furan (F9-F14) were bigger in size and showed some degree of aggregation

when compared with PMs containing chloroform: acetonitrile.

Scanning Electron Microscopy Images of Camptothecin Polymeric Micelles

Containing Chloroform: Acetonitrile

Fig 15: F3, (1:12.5) Fig 16: F4, (1:25)

Fig 17: F7, (1:12.5) Fig 18: F8, (1:25)

To investigate the morphology of prepared polymeric micelles SEM

observations were conducted. The SEM images (Fig 15-18) of PMs made of Me

PEG-b-PCL showed that the micelles were discrete and round in shape. In this

image, the aggregation of micelles was prevented effectively by the steric

repulsion effect of PEG shells (Yamamoto et al., 2001).


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Transmission Electron Microscopy Images of Camptothecin Polymeric

Micelles Containing Chloroform: Acetonitrile

Fig 19: F4, (1:25)

Fig 20: F8, (1:25)

The morphology and particle size evaluation of prepared CPT-PMs

containing F4 (PI, 1:25) and F8 (PII, 1:25) was further confirmed by TEM (Fig19 &

20). TEM images showed the uniform distribution of PMs. For formulation

containing chloroform: acetonitrile (F4, 1:25, PI) the particle size was found to be

1 m

1 m


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195.4 nm and for F8 (1:25, PII) it was 170.5 nm. The TEM showed smaller size

than that observed by dynamic light scattering examination due to the shrinking

of their relatively thick PEG shell in the drying process. It was also observed that

increasing the molecular weight of PCL increased the particle size; however,

increasing the PEG chain length with similar PCL chain length produced a

smaller particle size (F5-F8 (PI) and F12-F14 (PII)). This is because PEG was

able to moderate the association of the copolymer molecules during the formation

of the particles (Masato Watanabe et al., 2005).

6.4 ENCAPSULATION EFFICIENCY DETERMINATION

Table 9: Encapsulation Efficiency of CPT Loaded PMs

Using Different Drug: Polymer Ratio

Formulation code Drug : polymer ratio % Drug entrapped

F1 1:2.5 60.30 ± 0.6

F2 1:5 78.02 ± 0.5

F3 1:12.5 82.75 ± 0.5

F4 1:25 83.80 ± 0.8

F5 1:2.5 56.21 ± 0.8

F6 1:5 77.89 ± 0.7

F7 1:12.5 79.50 ± 0.5

F8 1:25 82.00 ± 0.4

Values represents ± SD, n=3


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Table 10: Encapsulation Efficiency of CPT Loaded PMs

Using Different Drug: Polymer Ratio

Formulation code Drug : polymer ratio % Drug entrapped

F9 1:5 41.53 ± 1.2

F10 1:12.5 45.61 ± 0.9

F11 1:25 51.03 ± 0.7

F12 1:5 38.64 ± 1.1

F13 1:12.5 44.54 ± 0.6

F14 1:25 48.19 ± 0.5

Values represents ± SD, n=3

Data Inference from Tables 9 and 10

Encapsulation efficiency was determined as per the method described in

section 5.11.

Several factors may affect the drug encapsulation efficiency of the

polymeric micelles prepared by emulsification solvent evaporation method. The

factors are,

a) the affinity of the loaded drug for the core-forming polymer

b) the volume of the hydrophobic core

c) Drug-drug interaction (i.e., its ability to self-aggregate) (Praneet

Opanasopit et al., 2004).

Among these three, the compatibility between the drug and the core-

forming block is said to be the main factor.


84


In order to obtain PMs with high drug loading, formulations containing

14 different ratios of drug: polymer ratio was prepared. For polymeric micelles

prepared using chloroform: acetonitrile, the highest encapsulation efficiency of

83.8 ± 0.8% was obtained for PMs (F4) containing 1:25 ratio of PI and

82.0 ± 0.4% for PMs (F8) containing 1:25 ratio of PII. For PMs prepared using

THF (F9-F14), the encapsulation efficiency was very low and ranged from 41.53

± 1.2% to 51.03 ± 0.7% (Table 9 and 10).

6.4.1 Effect of Copolymer Concentration on the Entrapment Efficiency

In this study, the PMs prepared with a 1:25 ratio of block copolymer I (F4)

showed higher encapsulation efficiency than PMs prepared with 1:5 and 1:12.5

ratios of the same polymer. However, it was found that a drug /copolymer feeding

ratio higher than 1:25 caused a large amount of particle aggregation and very low

polymeric micelle yield (data not shown). This is important because the state of

the incorporated drug in the PMs is the primary factor that determines the release

profile of a drug delivery system. The PMs prepared with higher copolymer

concentrations yielded higher encapsulation efficiencies. Our results are similar to

the results stated by Bin Shi et al., 2005. Higher copolymer concentrations would

accelerate the solidification of PMs, which could in turn retain the drug in the

polymer matrix more effectively.

6.4.2 Effect of Organic Solvent on Drug Entrapment Effeciency

When the organic solvent and the feed weight ratio were fixed, the loading

efficiency increased as the content of hydrophobic PCL block in the copolymer

increased (Hamidreza Montazeri Aliabadi et al., 2007). When chloroform:

acetonitrile was used as solvent, higher encapsulation efficiency was obtained

(F1-F8) than those obtained using THF (F9-F14) for all the block co polymers.

Therefore, chloroform: acetonitrile is a more favorable solvent for CPT than THF

when emulsification solvent evaporation method was used (Sang Cheon

Lee et al., 2003).


85


The entrapment of hydrophobic drugs in polymeric micelles is driven by

the hydrophobic interactions between the drug and hydrophobic segments of

polymers. The result obtained also implies that the increase in the hydrophobic

chain length of block copolymer enhances an interaction between CPT, which is

a hydrophobic drug and polycaprolactone, leading to an increase in the drug

loading content.

6.5 CRITICAL MICELLAR CONCENTRATION (CMC) OF CAMPTOTHECIN

POLYMERIC MICELLES

Table 11: Critical Micellar Concentration of CPT-PMs

Containing Chloroform: Acetonitrile

Formulation code Drug : polymer ratio CMC (µg/ml)

F1 1:2.5 18

F2 1:5 10

F3 1:12.5 13

F4 1:25 5

F5 1:2.5 32

F6 1:5 28

F7 1:12.5 20

F8 1:25 12


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Table 12: Critical Micellar Concentration of CPT-PMs

Containing Tetra hydro Furan

Formulation code Drug : polymer ratio CMC (µg/ml)

F9 1:5 34

F10 1:12.5 31

F11 1:25 26

F12 1:5 42

F13 1:12.5 40

F14 1:25 38

Data Inference from Tables 11 and 12

Critical micellar concentration of prepared polymeric micelles was carried

out as per the procedure specified in section 5.12.

The stability of micelles in vitro depends on the CMC values. The stability

is a very important parameter in the drug delivery application of polymeric

micelles because intravenous injections of micellar solutions are associated with

extreme dilutions by circulating blood. If the concentration of a micelle forming

polymer in the circulation drops below the CMC, the micelles may be prematurely

destroyed resulting in the release of encapsulated drug into the circulation before

it reaches its target; this could be dangerous because a poorly soluble drug may

precipitate inside blood vessels. Upon dilution to CMC, micelles begin to

dissociate into unimers and thus release drugs. The lower the CMC value, the

better the micelle stability (Tatsuhiro Yamamoto et al., 2007).

CMC of block copolymers were analyzed using pyrene as a fluorescent

probe. Pyrene is known to exhibit a peak shift in its excitation spectrum upon its

incorporation into a hydrophobic inner-core. In the presence of micelles, pyrene

is transferred from the aqueous environment to the non polar region of the micelle

interior core, which results in significant spectroscopic changes of pyrene

(Bin Shi et al., 2005). In this study the CMC of F4 (PI, 1:25, chloroform:

acetonitrile) and F8 (PII, 1:25, chloroform: acetonitrile) were found to be 5 and


87


12µg/ml respectively, whereas for F11 (PI, 1:25, THF) and F14 (PII, 1:25, THF) it

was found to be 26 and 38µg/ml respectively. The polymeric micelles prepared

using chloroform: acetonitrile showed decrease in CMC than the micelles

containing THF. Also the CMC decrease with an increase in the length of PCL

core block (F1-F4 and F9-F11), in which it has been shown that a larger lipophilic

area facilitates and stabilizes micellar formation (Bin Shi et al., 2005).

These results showed that the chain length and nature of the hydrophobic

block has the most profound effects (Allen et al., 1999) on critical micellar

concentration and it is primarily controlled by the length of a hydrophobic block.

The longer the length, the lower the CMC. The incorporation of hydrophobic

drugs in micelle cores may decrease the CMC (Natalya Rapoport et al., 2007).

The CMC is less sensitive to the length of a hydrophilic block.

Also these results on experimental CMC values vs hydrophobic block

length coincide well with those reports given by Astafieva et al. They reported

that the onset of micellization was determined mainly by the nature and the

length of the hydrophobic block, where the nature of the hydrophilic block had

only a slight dependence on the onset of micellization.


88


6.6 IINN VVIITTRROO RREELLEEAASSEE SSTTUUDDIIEESS

TThhee iinn vviittrroo rreelleeaassee ssttuuddiieess wweerree ccaarrrriieedd oouutt bbyy ddiiaallyyssiiss bbaagg ddiiffffuussiioonn

tteecchhnniiqquuee ssppeecciiffiieedd iinn sseeccttiioonn 55..1133..

TTaabbllee 1133:: IInn VViittrroo RReelleeaassee PPrrooffiillee ooff CCaammppttootthheecciinn SSoolluuttiioonn

Time in hours *Absorbance *Concentration

(mg/ml) *Cumulative % drug released

1 0.287 0.215 35.962 ± 0.2

2 0.375 0.281 46.999 ± 0.1

3 0.441 0.330 55.053 ± 0.5

4 0.554 0.414 69.038 ± 0.2

5 0.698 0.522 87.137 ± 0.5

6 0.775 0.579 96.600 ± 0.6

* EEaacchh vvaalluuee wwaass aann aavveerraaggee ooff tthhrreeee ddeetteerrmmiinnaattiioonnss;; VVaalluueess aarree eexxpprreesssseedd aass mmeeaann ±±SSDD

TTaabbllee 1144:: IInn VViittrroo RReelleeaassee ffrroomm PPoollyymmeerriicc MMiicceelllleess ((FF11)) CCoonnttaaiinniinngg

CChhlloorrooffoorrmm:: AAcceettoonniittrriillee ((PPII,, DDrruugg:: PPoollyymmeerr RRaattiioo 11::22..55))



1 0.146 0.110 30.429 ± 0.2

2 0.157 0.118 32.685 ± 0.8

3 0.171 0.129 35.813 ± 0.6

4 0.191 0.144 39.952 ± 0.9

5 0.198 0.149 41.208 ± 0.5

6 0.220 0.165 45.638± 0.1

7 0.246 0.184 50.904±0.7

8 0.261 0.196 54.190 ± 0.5

9 0.289 0.217 60.143± 0.9

10 0.325 0.244 67.490 ± 0.8

11 0.359 0.269 74.618 ± 0.8

12 0.386 0.289 79.884 ± 0.6

24 0.425 0.318 88.115 ± 0.7

36 0.459 0.344 95.233 ± 0.9 * EEaacchh vvaalluuee wwaass aann aavveerraaggee ooff tthhrreeee ddeetteerrmmiinnaattiioonnss;; VVaalluueess aarree eexxpprreesssseedd aass mmeeaann ±±SSDD


89



CChhlloorrooffoorrmm:: AAcceettoonniittrriillee ((PPII,, DDrruugg:: PPoollyymmeerr RRaattiioo 11::55))



1 0.104 0.079 17.124 ± 0.5

2 0.119 0.089 19.429 ± 0.8

3 0.129 0.097 20.952 ± 0.5

4 0.146 0.109 23.731 ± 0.6

5 0.128 0.096 26.904 ± 0.2

6 0.198 0.148 32.078 ± 0.9

7 0.248 0.186 40.286 ± 0.3

8 0.272 0.204 44.065 ± 0.6

9 0.300 0.224 48.537 ± 0.2

10 0.325 0.243 52.533 ± 0.9

11 0.344 0.258 55.912 ± 0.3

12 0.377 0.282 60.947 ± 0.9

24 0.501 0.375 81.157 ± 0.8

36 0.545 0.408 88.315±0.1

*EEaacchh vvaalluuee wwaass aann aavveerraaggee ooff tthhrreeee ddeetteerrmmiinnaattiioonnss;; VVaalluueess aarree eexxpprreesssseedd aass mmeeaann ±±SSDD


90



CChhlloorrooffoorrmm:: AAcceettoonniittrriillee ((PPII,, DDrruugg:: PPoollyymmeerr RRaattiioo 11::1122..55))



1 0.129 0.097 19.547 ± 0.9

2 0.159 0.120 24.193 ± 0.7

3 0.187 0.141 28.593 ± 0.4

4 0.206 0.155 31.372 ± 0.8

5 0.254 0.191 38.591±0.5

6 0.281 0.211 42.671 ±0.9

7 0.292 0.219 44.228 ± 0.5

8 0.307 0.230 46.367 ± 0.2

9 0.312 0.234 47.226 ± 0.9

10 0.317 0.237 47.877 ± 0.4

11 0.327 0.245 49.445 ± 0.6

12 0.358 0.268 54.000 ± 0.8

24 0.498 0.372 75.022 ± 0.5

36 0.548 0.410 82.613±0.1



91



CChhlloorrooffoorrmm:: AAcceettoonniittrriillee ((PPII,, DDrruugg:: PPoollyymmeerr RRaattiioo 11::2255))


(mg/ml) * Cumulative % drug released

1 0.072 0.055 10.939 ± 0.2

2 0.080 0.061 12.322 ± 0.9

3 0.096 0.072 14.412 ± 0.2

4 0.110 0.083 16.586 ± 0.4

5 0.139 0.105 20.966 ± 0.4

6 0.169 0.127 25.395 ± 0.1

7 0.200 0.150 29.962 ± 0.4

8 0.212 0.159 31.739 ± 0.8

9 0.23 0.174 34.773 ± 0.1

10 0.25 0.190 37.947 ± 0.3

11 0.269 0.202 40.247 ± 0.2

12 0.33 0.247 49.228 ± 0.2

24 0.466 0.349 69.513 ± 0.4

36 0.514 0.385 76.581 ± 0.9



92


Fig 21: Comparative In Vitro Release Profiles of Camptothecin from

Polymeric Micelles (F1, F2, F3 & F4)


93



CChhlloorrooffoorrmm:: AAcceettoonniittrriillee ((PPIIII,, DDrruugg:: PPoollyymmeerr RRaattiioo 11::22..55))



1 0.146 0.110 32.731 ± 0.8

2 0.208 0.156 34.286 ± 0.5

3 0.170 0.128 37.989± 0.9

4 0.182 0.137 40.661 ± 0.8

5 0.198 0.149 44.183 ± 0.6

6 0.213 0.160 47.567 ± 0.6

7 0.234 0.176 52.346 ± 0.4

8 0.256 0.192 56.997 ± 0.9

9 0.273 0.205 60.392± 0.3

10 0.297 0.223 66.309 ± 0.8

11 0.340 0.255 75.739 ± 0.9

12 0.360 0.270 80.261 ± 0.4

24 0.394 0.295 87.631 ± 0.9

36 0.433 0.324 96.113 ± 0.8



94



CChhlloorrooffoorrmm:: AAcceettoonniittrriillee ((PPIIII,, DDrruugg:: PPoollyymmeerr RRaattiioo 11:: 55))



1 0.130 0.098 21.062 ± 0.4

2 0.151 0.114 24.585 ± 0.9

3 0.175 0.132 28.311 ± 0.5

4 0.210 0.158 33.864 ± 0.6

5 0.246 0.184 39.465 ± 0.5

6 0.281 0.211 45.241 ± 0.7

7 0.304 0.228 48.892 ± 0.4

8 0.316 0.237 50.892 ± 0.2

9 0.32 0.244 52.321 ± 0.8

10 0.344 0.258 55.228 ± 0.7

11 0.378 0.283 60.575±0.9

12 0.419 0.314 67.226 ± 0.2

24 0.55 0.414 88.601 ± 0.9

36 0.587 0.439 94.015 ± 0.3



95



CChhlloorrooffoorrmm:: AAcceettoonniittrriillee ((PPIIII,, DDrruugg:: PPoollyymmeerr RRaattiioo 11:: 1122..55))



1 0.124 0.093 19.595 ± 0.5

2 0.147 0.111 23.374 ± 0.8

3 0.187 0.141 29.769 ± 0.9

4 0.224 0.168 35.372 ± 0.7

5 0.254 0.191 40.196 ± 0.4

6 0.275 0.206 43.321 ± 0.8

7 0.292 0.219 46.052 ± 0.3

8 0.313 0.235 49.445 ± 0.9

9 0.332 0.249 52.304 ± 0.7

10 0.339 0.254 53.349 ± 0.2

11 0.35 0.262 55.045 ± 0.5

12 0.384 0.288 60.585 ± 0.7

24 0.536 0.401 84.114 ± 0.9

36 0.575 0.430 90.315 ± 0.5



96



CChhlloorrooffoorrmm:: AAcceettoonniittrriillee ((PPIIII,, DDrruugg:: PPoollyymmeerr RRaattiioo 11:: 2255))



1 0.098 0.074 15.183 ± 0.9

2 0.159 0.12 24.567 ± 0.8

3 0.171 0.129 26.395 ± 0.5

4 0.201 0.151 30.737 ± 0.2

5 0.210 0.158 32.261 ± 0.6

6 0.230 0.173 35.294 ± 0.5

7 0.271 0.203 41.294 ± 0.7

8 0.284 0.213 43.374 ± 0.6

9 0.30 0.232 47.166 ± 0.8

10 0.321 0.241 49.025 ± 0.4

11 0.338 0.253 51.593 ± 0.9

12 0.36 0.272 55.445 ± 0.8

24 0.514 0.385 78.413 ± 0.7

36 0.587 0.439 89.361 ± 0.7



97


Fig 22: Comparative In Vitro Release Profiles Of CPT from Polymeric

Micelles (F5, F6, F7 & F8)


98



TTeettrraahhyyddrroo FFuurraann ((PPII,, DDrruugg:: PPoollyymmeerr RRaattiioo 11:: 55))



1 0.123 0.093 37.413± 0.8

2 0.134 0.101 40.694± 0.9

3 0.145 0.109 43.779± 0.6

4 0.158 0.119 47.886± 0.7

5 0.170 0.128 51.419± 0.5

6 0.181 0.136 54.645± 0.4

7 0.198 0.149 59.919± 0.3

8 0.214 0.161 64.846± 0.3

9 0.230 0.173 69.775± 0.8

10 0.250 0.188 75.818± 0.9

11 0.265 0.199 80.001±0.9

12 0.28 0.212 85.412± 0.7

24 0.320 0.240 96.661 ± 0.5



99



TTeettrraahhyyddrroo FFuurraann ((PPII,, DDrruugg:: PPoollyymmeerr RRaattiioo 11:: 1122..55))



1 0.129 0.097 35.617± 0.5

2 0.143 0.108 39.547 ± 0.7

3 0.162 0.122 41.101± 0.6

4 0.165 0.124 45.614± 0.8

5 0.178 0.134 49.313± 0.5

6 0.190 0.143 52.345± 0.8

7 0.213 0.160 58.678± 0.4

8 0.218 0.164 60.212± 0.9

9 0.238 0.179 65.719± 0.9

10 0.267 0.200 73.141± 0.8

11 0.284 0.213 78.117± 0.6

12 0.293 0.220 80.513± 0.7

24 0.338 0.253 92.629 ± 0.8



100



TTeettrraahhyyddrroo FFuurraann ((PPII,, DDrruugg:: PPoollyymmeerr RRaattiioo 11:: 2255))



1 0.137 0.103 33.718± 0.9

2 0.157 0.118 38.635± 0.9

3 0.162 0.127 41.667± 0.5

4 0.181 0.136 44.495± 0.4

5 0.198 0.149 48.667± 0.6

6 0.213 0.160 52.431± 0.7

7 0.24 0.182 59.575 ± 0.9

8 0.250 0.188 61.641± 0.7

9 0.26 0.197 64.432± 0.9

10 0.292 0.219 71.845± 0.5

11 0.311 0.233 76.149± 0.9

12 0.323 0.242 79.317± 0.9

24 0.368 0.276 90.449± 0.8



101


Fig 23: Comparative In Vitro Release Profiles of CPT from Polymeric

Micelles (F9, F10 and F11)


102



TTeettrraahhyyddrroo FFuurraann ((PPIIII,, DDrruugg:: PPoollyymmeerr RRaattiioo 11:: 55))



1 0.124 0.094 40.661± 0.4

2 0.130 0.098 42.339±0.8

3 0.139 0.105 45.649± 0.9

4 0.155 0.117 50.745± 0.5

5 0.171 0.129 55.984± 0.6

6 0.185 0.139 60.013± 0.5

7 0.198 0.149 64.997± 0.7

8 0.216 0.162 69.881± 0.4

9 0.228 0.171 74.134± 0.2

10 0.245 0.184 79.447± 0.8

11 0.264 0.198 85.641± 0.7

12 0.280 0.210 90.910±0.9

24 0.297 0.223 96.343± 0.2



103



TTeettrraahhyyddrroo FFuurraann ((PPIIII,, DDrruugg:: PPoollyymmeerr RRaattiioo 11:: 1122..55))



1 0.142 0.107 40.196± 0.5

2 0.154 0.116 43.479± 0.8

3 0.157 0.118 44.513± 0.9

4 0.173 0.130 48.661± 0.7

5 0.193 0.145 54.419± 0.4

6 0.213 0.160 59.984± 0.8

7 0.230 0.173 64.781± 0.3

8 0.252 0.189 70.864± 0.9

9 0.263 0.197 73.918± 0.7

10 0.276 0.207 77.669± 0.2

11 0.296 0.222 83.119± 0.5

12 0.315 0.236 88.671± 0.7

24 0.338 0.253 94.918± 0.9



104



TTeettrraahhyyddrroo FFuurraann ((PPIIII,, DDrruugg:: PPoollyymmeerr RRaattiioo 11:: 2255))



1 0.147 0.111 38.413± 0.9

2 0.155 0.117 40.467± 0.8

3 0.165 0.124 43.199± 0.5

4 0.178 0.134 46.601± 0.2

5 0.204 0.153 53.119± 0.6

6 0.229 0.172 59.648± 0.5

7 0.240 0.180 62.567± 0.7

8 0.264 0.198 68.495± 0.6

9 0.276 0.207 71.639± 0.8

10 0.291 0.218 75.515 ± 0.4

11 0.311 0.233 80.868± 0.9

12 0.331 0.248 85.919± 0.8

24 0.359 0.269 93.317± 0.7



105


Fig 24: Comparative In Vitro Release Profiles of CPT from Polymeric

Micelles (F12, F13 and F14)

DDaattaa IInnffeerreennccee

The release behavior of a lipophilic compound from the core shell of

structured PMs is largely dependent on the hydrophobic property of the inner

core. The other factors influencing rate of drug release include:

a) The release media used.

b) The characteristic of the copolymer.

c) The particle size and the internal structure of the micelles.

d) The form of the drug loaded in the micelles.

The release rate of camptothecin from polymeric micelles seems to

decrease with an increase in molecular weight of Me PEG-b-PCL block

copolymer. The micelles composed of copolymer with long hydrophobic chains

would form large hydrophobic cores because of strong hydrophobic interaction

and the incorporated drug would take relatively long time to diffuse across the

polymer matrix to the medium. (Gref et al., 1994).


106


Phosphate-buffered saline, pH 7.4, was used as the release medium in

order to investigate the other factors concerned. In vitro release profiles of all the

formulation were compared with CPT solution.

In our study, CPT-PMs prepared using chloroform: acetonitrile containing

1:5 ratio of PI exhibited a burst release of 17.124 ± 0.5% in the first hour. For

PMs containing 1:12.5 ratio of PI it was 19.547 ± 0.9% and for PMs containing

1:25 ratio of block copolymer it was 10.939 ± 0.2%. For PII, PMs containing 1:5,

1:12.5 and 1:25 ratios of block copolymer, the burst release was 21.062 ± 0.4%,

19.595 ± 0.5 % and 15.183 ± 0.9% respectively. For the PMs prepared using

THF, the initial burst release was found to be 33-40% which was much higher

than the formulations containing chloroform: acetonitrile (10-32%).

The maximum percentage of drug (96.6 ± 0.6%) was released within 6

hrs for a solution of pure CPT. For the PM formulations of PI containing

chloroform:acetonitrile, however, the release was found to be 45.638± 0.1(F1),

32.078 ± 0.9% (F2), 42.671 ± 0.9% (F3) and 25.395 ± 0.1% (F4) at 6 hrs. For the

formulations containing PII (F5, F6, F7, F8), the percent released within 6 hrs was

47.567± 0.6, 45.241 ± 0.7%, 43.321 ± 0.8% and 35.294 ± 0.5% respectively

(Figures 21 and 22).

About 88.31 ± 0.1% of the total loaded drug was released from PMs

containing 1:5 ratio of block copolymer (PI) in 36 hrs. For the 1:12.5 ratio of block

copolymer I, it was found to be 82.613 ± 0.1% and 78.58 ± 0.1% for PMs

containing a 1:25 ratio.

For PMs containing 1:5, 1:12.5 and 1:25 ratios of P II, the total CPT

released in 36 hrs was 94.015 ± 0.3, 90.315 ± 0.5 & 89.36 ± 0.7% respectively.

For formulations prepared using THF as a solvent, the release rate was faster

than formulations prepared using chloroform: acetonitrile. For formulations

F9, F10 and F11containing PI the release was found to be 96.66 ± 0.5%,


107


92.62 ± 0.8% and 90.44 ± 0.8% respectively within 24 hrs. For formulations F12,

F13 and F14containing PII the release was 96.34 ± 0.2%, 94.91 ± 0.9% and

93.31 ± 0.7% respectively within 24 hrs (Fig 23 and 24).

The data showed that the release rate decreased as the diblock

copolymer ratio increased. PMs with larger size have a slower release rate.

Larger polymeric micelles have bigger hydrophobic cores and smaller surface

area, so it takes longer time for the incorporated drug to diffuse across the

polymer matrix to the particle surface and finally into the aqueous medium,

resulting in slower release.

These results also show that the longer the PCL chain length, the slower

the drug release. The longer chain length of PCL could result in larger particle

size, smaller surface area and greater diffusion layer thickness which slow down

the drug release. The slow steady release of CPT observed with all these

preparations demonstrates that the drug is more effectively encapsulated by ESE

method. In addition, the presence of PCL can increase the interaction between

the polymer matrix and the loaded drug and further slow the release rate. Similar

results were reported by Leyang zhang et al., 2007.

These reports showed that all samples exhibited a burst release of CPT at

the initial stage. This may be because a portion of the drug was deposited at the

region near or within the PEG shell and could gain access to aqueous medium

without the need of a long diffusion time (Yuan-Chia Chang et al., 2008). After the

initial burst, the CPT release rate slowed down and became steady in a sustained

release manner.

All block copolymer chains prepared by the emulsification solvent

evaporation method containing higher molecular weight PCL (13,000) with

chloroform: acetonitrile as a solvent showed slow release for a prolonged period

of time when compared to PMs containing THF.


108


Drug Release Kinetics

Table 28: Determination of Order of Release of Camptothecin from Polymeric Micelles

Formulation

Higuchi Korsmeyer

Peppas Zero order First order

Hixson-Crowell

Release mechanism

r2 r2 n

value r2 k0( h

-1) r2 K1 ( h-1) r2

n value

F4 0.997 0.993 0.246 0.966 4.04 0.994 0.141 0.997 0.141 FD

F8 0.994 0.998 0.312 0.984 4.98 0.991 0.122 0.996 0.133 FD

F11 0.990 0.996 0.288 0.961 6.88 0.997 0.098 0.998 0.145 FD

F14 0.993 0.994 0.291 0.984 7.45 0.989 0.112 0.996 0.125 FD

FD-Fickian diffusion


109


Data inference

Camptothecin polymeric micelles were prepared by ESE method using

diblock copolymers to retard drug release and to achieve the required release

profile. To study the release kinetics, data obtained from in vitro release studies

were plotted in various kinetic models viz., zero order, first order, Higuchi’s plot,

Korsmeyer-Peppas and Hixson-Crowell plots (Ju RTC et al., 1995) and the

results obtained were shown in table 28.

The formulations (F4, F8, F11 and F14) with the highest correlation

coefficient were shown first order and obey first order kinetics. It was found that

the in vitro release of CPT from PMs was best explained by Higuchi’s equation.

The drug release was proportional to the square root of time, indicating that the

drug release from the micelles was diffusion controlled. The data obtained were

also put in Korsmeyer-Peppas model in order to find out n value, which describes

the drug release mechanism and the drug release from all the formulations were

diffusion controlled. Hixson – Crowell plot showed that the release of drug from

polymeric micelles followed analomous diffusion (Abbas et al., 2006).


110


IN VIVO STUDIES

Since in vitro studies of the formulations containing chloroform:acetonitrile

showed smaller particle size, low polydispersity index, higher entrapment

efficiency and slower drug release, further in vivo studies were carried out for the

formulations F4 (PI, 1:25, chloroform:acetonitrile) and F8 (PII, 1:25,

chloroform:acetonitrile).

6.7 ANTICANCER ACTIVITY

In vivo anticancer studies were carried out in mice bearing Ehrlich Ascites

Carcinoma (EAC) / Daltons Lymphoma Ascites (DLA) cells as per the procedure

mentioned in section 5.14

Table 29: Effect of Camptothecin Polymeric Micelles on the Hematological

Parameters of Mice Bearing DLA

Treatment Total WBC

(cells/ml ×103)

RBC (million/ cu.mm)

Haemoglobin (Hb) (gm/dl)

Platelets (lakhs/Cu.mm)

Normal control

10.20 ± 0.20 4.5 ± 0.28 11.26 ± 0.30 2.58 ± 0.21

Tumour control

16.16 ± 0.40 2.11 ± 0.11 6.40 ± 0.09 1.62 ± 0.17

CPT solution 13.42 ± 0.24 2.81 ± 0.30 8.9 ± 0.18 1.82 ± 0.36

F4 (2 mg/kg) 11.95 ± 0.22 3.42 ± 0.23 9.6 ± 0.12 2.02 ± 0.38

F4 (4 mg/kg) 11.63 ± 0.21 3.68 ± 0.85 9.9 ± 0.28 2.11 ± 0.65

F4 (8 mg/kg) 11.08 ± 0.20 3.82 ± 0.36 10.4 ± 0.22 2.28 ± 0.54

F8 (2 mg/kg) 12.26 ± .26 3.28 ± 0.26 9.2 ± 0.19 1.99 ± 0.26

F8 (4 mg/kg) 12.12 ± 0. 26 3.34 ± 0.92 9.4 ± 0.52 2.08 ± 0.35

F8 (8 mg/kg) 11.73 ± 0. 19 3.45 ± 0.56 10.1 ± 0.66 2.19 ± 0.36

Datas are expressed in mean ± SD (n=6).


111


Fig 25: Effect of Camptothecin Polymeric Micelles on Total WBC Count

Values were expressed in mean ± SD (n=6). ** p< 0.01, *p<0.05 when compared with CPT

solution (ANOVA with post hoc Dunnett's test).


112


Fig 26: Effect of Camptothecin Polymeric Micelles on Total RBC Count

Values are expressed as mean ± SD (n=6). ** p< 0.01, *p<0.05 when compared with CPT solution

(ANOVA with post hoc Dunnett's test).


113


Fig 27: Effect of Camptothecin Polymeric Micelles on

Haemoglobin (Hb) Content




114


Fig 28: Effect of Camptothecin Polymeric Micelles on Platelets Count


(ANOVA with post hoc Dunnett’s test).


115


Data inference

Haematological parameters of tumour bearing mice showed significant

changes when compared to camptothecin solution. The Total WBC count, RBC,

Platelets and Hb content showed significant difference in CPT polymeric micelles

(F4 (PI, 1:25) and F8 (PII, 1:25) ) when compared to CPT solution. The reversal

of Hb content, RBC, platelet & WBC by camptothecin polymeric micelles

treatment towards the value of normal group clearly indicate that camptothecin

and its formulation have a protective action on haematopoietic system

(Table 29).

When compared with CPT solution and F8 (PII, 1:25,

chloroform:acetonitrile), CPT PMs containing F4 (PI, 1:25, chloroform:acetonitrile)

at 8mg/kg significantly decreases the total WBC count and significantly increases

the RBC, Platelets and Hb content (p<0.01).

Table 30: Effect of Camptothecin Polymeric Micelles on the Life Span, Body

Weight and Cancer Cell Count of Mice Bearing DLA

Treatment % increase in life span

(% ILS)

Increase in body weight (gms)

Cancer cell count (ml ×106)

Normal control ≥ 30days 1.22 ± 0.06 0

Tumour control 40% 8.46 ± 1.8 2.20 ± 0.22

CPT solution 59% 2.76 ± 0.22 1.09 ± 0.25

F4 (2 mg/kg) 78% 2.80 ± 0.29 0.95 ± 0.25

F4 (4 mg/kg) 83% 2.41 ± 0.33 0.91 ± 0.29

F4 (8 mg/kg) 88% 1.99 ± 0.52 0.87 ± 0.36

F8 (2 mg/kg) 67% 3.58 ± 0.22 1.01 ± 0.19

F8 (4 mg/kg) 73% 3.12 ± 0.66 0.96 ± 0.42

F8 (8 mg/kg) 80% 2.96 ± 0.39 0.91 ± 0.44

Datas are expressed in mean ± SD (n=6).


116


Fig 29: Effect of Camptothecin Polymeric Micelles on the Life Span

of Mice Bearing DLA

Values are expressed as mean ± SD, n= 6, *p<0.05,**p<0.01, when compared with CPT solution

(ANOVA with Post hoc Dunnett’s test).

Data Inference

The effect of camptothecin and its formulation on the survial of DLA

tumour bearing mice is shown in Fig 29.

The reliable criteria for judging the value of anti cancer drug is the

prolongation of life span of animal. In this study an increase in life span was

observed with treated groups. The DLA bearing mice administered with

camptothecin and its formulation at different doses (2mg, 4mg and 8mg/kg)

showed significant increase in average life span compared to the animals treated

with CPT drug solution given at a dose of 8mg/kg. The animals treated with

8mg/kg of CPT-PMs containing F4 (PI, 1:25, chloroform:acetonitrile) showed

highest significance (p<0.01) when compared with CPT solution and the % ILS

was found to be around 88% and for F8 (PII, 1:25, chloroform:acetonitrile) it was

found to be 80% (p<0.01).


117


When CPT-PMs were used, the reason of increased survivability might be

due to the enhanced vascular permeability and retention effect of polymeric

micelles. It was reported that higher molecular weight copolymers showed more

effectiveness in the tumour accumulation of polymers (Jubo Liu et al., 2006).

Fig 30: Effect of Camptothecin Polymeric Micelles on Increase in

Body Weight



Data inference

The effect of camptothecin and its formulation on the change in body

weight in tumour bearing mice is shown in figure 30.

The percentage increase in body weight was found to be significantly less

(p<0.01 for F4, 8mg/kg and p<0.05 for F8, 8mg/kg) in camptothecin polymeric

micelles in treated mice than CPT solution, indicating the anticancer nature of

camptothecin polymeric micelles.


118


All groups treated with CPT-PMs (2mg, 4mg, 8mg/kg) yielded significant

changes in the percentage increase in life span, reduction in tumour volume,

change in body weight of tumour bearing mice when compared with free CPT

solution. The group treated with CPT loaded PMs (F4, PI, 1:25,

chloroform:acetonitrile) at 8mg/kg showed the highest significance when

compared with F8 (PII, 1:25, chloroform:acetonitrile).

Fig 31: Effect of Camptothecin Polymeric Micelles on the

Cancer Cell Count

Values are expressed as mean ± SD (n=6). ** p< 0.01, *p<0.05 when compared with CPT solution (ANOVA with post hoc Dunnett's test).


119


Fig 32: Viable Tumour Cell Count of Camptothecin Polymeric Micelles

Camptothecin Formulation (F4-2 mg/kg)






Camptothecin Solution Tumour Control


120


Data Inference

The microscopical views of the cell counts have been shown in the figure

32. The results of the cancer cell count indicate that the group treated with CPT

loaded PMs formulations (F4 & F8) significantly reduced the viable tumour cell

count (p<0.01) when compared to CPT solution (Table 30).

Table 31: Effect of Camptothecin Polymeric Micelles Formulation on

Serum Lipid Profile

Groups Total cholesterol Triglycerides (TGL)

Normal control 108.5 ± 2.62 129.95 ± 3.10

Tumour control 140.70 ± 3.65 210.2 ± 3.26

CPT solution 130.46 ± 2.81 176.22 ± 2.42

F4 (2 mg/kg) 129.78 ± 3.62 160.31 ± 2.18

F4 (4 mg/kg) 125.62 ± 3.46 154.46 ± 2.72

F4 (8 mg/kg) 112.38 ± 2.92 145.4 ± 3.05

F8 (2 mg/kg) 128.30 ± 2.22 166.30 ± 2.39

F8 (4 mg/kg) 126.40 ± 2.08 160.42 ± 2.52

F8 (8 mg/kg) 122.46 ± 3.10 157.66 ± 2.76

Datas are expressed as mean ± SD (n=6).


121


Fig 33: Effect of Camptothecin Polymeric Micelles Formulation on

Serum Lipid Profile – Total Cholesterol




122


Fig 34: Effect of Camptothecin Polymeric Micelles Formulation on

Serum Lipid Profile – Triglycerides

Values are expressed as mean ± SD (n=6). ** p< 0.01, *p<0.05 when compared with CPT

solution (ANOVA with post hoc Dunnett 's test).

Data inference

The levels of serum cholesterol and triglycerides of DLA control,

camptothecin drug solution and its polymeric micelles (F4 and F8) were shown in

table 31. The serum lipid profiles of DLA control animals were found to be higher

when compared to other CPT PMs treated groups. The triglyceride levels and

cholesterol levels were significantly lower (P<0.01) in the DLA bearing animals

in treated groups (F4, 1:25 and F8, 1:25) .


123


Table 32. Effect of Camptothecin Polymeric Micelles Formulation on Solid

Tumour Volume

Treatment 15th day 20th day 25th day 30th day

Tumour control

6.6 ± 0.64 8.28 ± 0.42 11.1 ± 0.38 12.05 ± 0.42

CPT solution 4.4 ± 0.36 5.48 ± 0.22 7.16 ± 0.18 7.18 ± 0.25

F4 (2 mg/kg) 3.5 ± 0.42 3.79 ± 0.15 3.86 ± 0.29 4.3 ± 0.52

F4 (4 mg/kg) 3.26 ± 0.56 3.5 ± 0.39 3.71 ± 0.25 4.01 ± 0.32

F4 (8 mg/kg) 2.21 ± 0.22 2.3 ± 0.22a 2.54 ± 0.22 2.94 ± 0.52

F8 (2 mg/kg) 3.81 ± 0.5 4.11 ± 0.48 4.71 ± 0.23 5.07 ± 0.12

F8 (4 mg/kg) 3.56 ± 0.38 3.91 ± 0.22 4.22 ± 0.46 4.6 ± 0.25

F8 (8 mg/kg) 3.21 ± 0.68 3.46 ± 0.4 3.8 ± 0.15 4.1 ± 0.42

Datas are expressed as mean ± SD (n=6).


124


Fig 35: Effect of Camptothecin Polymeric Micelles Formulation on Solid

Tumour Volume

Values are expressed as mean ± SD (n=6). *** p<0.001, ** p< 0.01, *p<0.05 when compared with

CPT solution (ANOVA with post hoc Dunnett test).

Data inference

The in vivo antitumour effect of CPT loaded polymeric micelles was

assessed using EAC / DLA bearing mice as the model animals. Table 32 lists the

increase in tumour volume of all the tested groups in mice bearing EAC.

PMs with smaller particle diameter and a PEG coated surface have been

found to well avoid entrapment by the RES and to well leak in diseased areas

with highly permeable blood vessels resulting in passive targeting to the diseased

states (Yokoyama et al., 1993, Kwon et al., 1994). This is known as the EPR

effect. In order to sufficiently acquire this EPR effect, we evaluated the

antitumour activity of CPT loaded micelles.


125


As shown in Fig 35 tumour growth in control mice was distinct and tumour

volume was rapidly increased. On the other hand tumour volume was

significantly suppressed by treatment of CPT loaded polymeric micelles when

compared to CPT solution. The polymeric micelles containing F4 (PI, 1:25,

chloroform:acetonitrile) showed highest reduction in tumour growth when

compared to polymeric micelles containing F8 (PII, 1:25, chloroform:acetonitrile).

It can be seen that CPT-PMs containing F4 (PI, 1:25, 8mg/kg) suppressed the

tumour growth more significantly (p<0.001) than free CPT. The PMs containing

F8 (PII, 1:25, 8mg/kg) showed less significance (p<0.01) when compared with

F4. This could be attributed to the enhanced permeation and retention effect of

nano sized micellar delivery systems.

Fast growing tumour tissues need a tremendous amount of oxygen and

nutrients supplied by blood vessels. They release special growth factors

including vascular endothelial cell growth factor (VEGF) to facilitate neo-

vascularization. As a result, many new vessels are formed, but their cell

junctions are not as tight as those normal tissues. CPT-PMs were likely to freely

pass through the endothelian junctions of the capillaries in tumour tissue, but not

in normal tissue. A combined effect of the passive targeting and enhanced

cellular uptake would be the main reason for the suppression of tumour growth

(Hyuk Sang Yoo et al., 2004).

The in vivo antitumour study indicated that compared with free CPT, CPT

loaded polymeric micelles showed a notable change in enhancing the antitumour

efficacy. This may be because of the sustained release characteristic of the CPT

loaded polymeric micelles which may result in prolonged exposure of the tumour

cells to the attack of the antitumour drug, which is essential for S-phase specific

drugs like CPT.


126


6.8 TISSUE DISTRIBUTION STUDIES

Tissue distribution studies of camptothecin polymeric micelles were

carried out in tumour bearing mice as per the procedure given in section 5.16

HPLC Chromatogram of Camptothecin in Spiked Plasma


127


Table 33: Standard Plot of Camptothecin in Spiked Plasma

S.No Concentration (µg/ml) Peak area

1 25 27888

2 50 55000

3 75 80999

4 100 109057

5 125 138876

Fig 41: Standard plot of Camptothecin in Spiked Plasma


128


HPLC Chromatogram of Camptothecin Solution


129


HPLC Chromatogram of F4 (PI, 1:25)


130


HPLC Chromatogram of F8 (PII, 1:25)


131


Table 34: Tissue Distribution of Camptothecin Drug Solution

in Various Organs

CPT in solution Concentration (µg/ml) % Drug distributed

Plasma 0.86 1.72 ± 0.2

Lungs 3.3 6.6 ± 0.7

Liver 5.9 11.8 ± 0.8

Spleen 7.3 14.6 ± 0.9

Kidney 4.1 8.2 ± 1.1

Tumour 0.99 1.98 ± 0.6

Data are given as mean ± SD (n=3)

Table 35: Tissue Distribution of Camptothecin Polymeric Micelles (F4, PI,

1:25, chloroform: acetonitrile) in Various Organs

F4 (PI, 1:25) Concentration (µg ml) % Drug distributed

Plasma 3.9 7.8 ± 1.1

Lungs 2.01 4.02 ± 0.9

Liver 0.76 1.52 ± 0.6

Spleen 0.97 1.94 ± 0.6

Kidney 3.46 6.92 ± 0.9

Tumour 10.5 21.0 ± 0.8



132


Table 36: Tissue Distribution of Camptothecin Polymeric Micelles (F8, PII,

1:25, chloroform: acetonitrile) in Various Organs

F8 (PII, 1:25) Concentration (µg ml) % Drug distributed

Plasma 3.3 6.60 ± 0.8

Lungs 2.3 5.75 ± 0.6

Liver 0.94 1.88 ± 0.9

Spleen 1.33 2.66 ± 0.5

Kidney 3.67 7.34 ± 0.8

Tumour 7.9 15.8 ± 0.9


Fig 60: Comparative Tissue Distribution of Camptothecin

Polymeric Micelles

CPT tissue distribution in mice bearing EAC cells 24hrs after i.v. injection of CPT loaded PMs (F4 & F8) and CPT solution at a dose of 2.5mg/kg. Each value represents the mean ±SD (n=3), **p<0.01, *p<0.05 compared with CPT solution. (One way ANOVA followed by post hoc Dunnett’s test)


133


Data inference

Fig 60 shows the tissue distribution profiles of CPT loaded polymeric

micelles and CPT solution after i.v. injection of 2.5mg/kg into mice bearing EAC.

Tissue distribution study was conducted in order to gain a deeper insight

into the in vivo behavior of the prepared CPT polymeric micelles, and to better

elucidate the reasons why drug loaded polymeric micelles had superior

antitumour efficacy than free CPT solution. Considering that, tissue distribution in

tumour bearing animals may be different from normal animals due to some

physiological changes brought about by tumour development, EAC bearing mice

instead of normal mice were employed in the tissue distribution investigations.

For both the formulations, rapid accumulation of copolymer in the various

organs was observed. Blood plasma levels of CPT loaded micelles containing F4

(PI, 1:25, chloroform: acetonitrile) and F8 (PII, 1:25, chloroform: acetonitrile) were

relatively higher (7.8 % and 6.60 % respectively) than CPT solution (1.72 %).

Liver showed significantly decreased uptake of CPT polymeric micelles

containing F4 and F8 (1.52 % and 1.88 % respectively) when compared to CPT

solution (11.8 %). Tumour accumulation of CPT loaded micelles of F4 and F8

was higher (21.0 % and 15.8 % respectively) than CPT solution (1.98 %). These

results showed that the PMs possess the ability to deliver large amounts of CPT,

in its most active form, to the tumour site by passive targeting with a long

circulating carrier (Table 35 & 36).

The CPT concentration in lungs (4.02% and 5.75% for F4 and F8

respectively) was also high possibly due to the filtration effect of the lung capillary

bed that removed some large particles or their aggregates. Concentration in

kidney, one of the major elimination pathways of the original CPT, was low and

did not show any significant difference between groups treated with CPT loaded

polymeric micelles. However the CPT concentration in liver, a major RES organ,


134


was significantly (p<0.01) lower, which might be attributed to the steric

stabilization provided by the PEG shell and could be viewed as a proof of the

RES evading ability of polymeric micelles. Micelles surface-modified with PEG

can stay in the circulation much longer, resulting in considerably high blood

concentration of loaded drug for a prolonged period of time and the passive

targeting effect.

When compared with camptothecin polymeric micelles containing F8

(PII, 1:25), F4 (PI, 1:25) showed highly significant results due to its longer

lipophilic chain length (PCL 13,000).

Therefore Me PEG-b-PCL polymeric micelles with smaller size seem to be

more capable of staying in circulation for a long period, which might be

advantageous in the delivery of antitumour drugs. The results obtained here

indicate that by utilizing the polymeric micelles as drug carriers, the blood

concentration of CPT could be maintained for a long period and the tissue

distribution could also be altered. An optimized tissue distribution may lead to

improved drug efficacy and reduced side effects.

66..99 PPHHAARRMMAACCOOKKIINNEETTIICC SSTTUUDDIIEESS

PPhhaarrmmaaccookkiinneettiicc ssttuuddiieess wweerree ccaarrrriieedd oouutt aass ppeerr tthhee pprroocceedduurree eexxppllaaiinneedd

iinn sseeccttiioonn 55..1177..


135


TTaabbllee 3377:: PPllaassmmaa DDrruugg CCoonncceennttrraattiioonn ooff CCaammppttootthheecciinn SSoolluuttiioonn aanndd

CCaammppttootthheecciinn PPoollyymmeerriicc MMiicceelllleess

Time ( hrs) Camptothecin drug in solution (ng/ml)

F 4 (PI, 1:25) (ng/ml)

F 8 (PII, 1:25) (ng/ml)

0.5 8.3 627 13.8

1 7.8 528 12.9

1.5 7.5 366 11.7

2 6.3 123 10.3

3 4.5 49 9.9

4 4.2 35.4 7.2

5 - 19.5 4.5

6 - 9.6 4.2

7 - 1.08 -

8 - - -

12 - - -


TTaabbllee 3388:: PPhhaarrmmaaccookkiinneettiicc PPaarraammeetteerrss ooff CCaammppttootthheecciinn SSoolluuttiioonn aanndd

CCaammppttootthheecciinn PPoollyymmeerriicc MMiicceelllleess

Parameters Drug in

solution (i.v) F4 (PI, 1:25)

(i.v)

F8 (PII, 1:25)

(i.v)

AUC 0-last

(ng/ml.h) 33.6 3519.3** 68.71*

t1/2 (hrs) 0.85 3.8** 3.3*

MRT(hrs) 0.9 5.5** 4.82*

Total clearance (ml/min/kg)

59.1 9.47** 37.3*

Vd (ml/kg) 692.5 1923.9** 1092.5*

Kel (min-1) 0.85 0.18* 0.21* Data are given as mean ± SD (n=3)

Pharmacokinetic parameters of CPT PMs and CPT solution following i.v. administration in rabbits. **denotes p<0.01, * denotes p<0.05 when compared with CPT solution (ANOVA followed by post hoc Dunnett’s test)


136


Data inference

The Me PEG-b-PCL micelles showed initial higher blood circulating levels

compared with free CPT. After 4 hrs, the free CPT had been removed from the

circulation and could not be detected. On the contrary, Me-PEG-b-PCL micelles

exhibited a remarkably delayed blood clearance and the concentration of CPT of

MePEG5000-b-PCL13, 000 (F4, 1:25) and MePEG5000-b-PCL5000 (F8, 1:25) in blood

at 4 hrs after i.v.administration was about 35.4 ng / ml and 7.2 ng/ ml respectively

(Table 38).

The concentration time- curves for Me PEG-b-PCL micelles and free CPT

in rabbits were fitted by the non compartmental model. The results showed that

Me PEG-b-PCL micelles with PEG molecular weight of 5000 could extend the

half-life of CPT to 3.8 hrs and 3.3 hrs [F4 (PI, 1:25, chloroform:acetonitrile) and

F8 (PII, 1:25, chloroform:acetonitrile) respectively].

The in vivo characteristics of Me-PEG-b-PCL micelles were consistent

with the micelles in vitro physicochemical characteristics. The longer PCL chain

length would lead to the reduction of CMC value, the increase of drug loading,

stabilized CPT and slower drug release patterns. The longer PEG chain length

would lead to less negative zeta potential. These physicochemical parameters

contributed together to the in vivo pharmacokinetic characteristics of Me PEG-b-

PCL micelles.

Since Me-PEG5000-b-PCL13, 000 (F4, 1:25) micelles had longer PCL chain

length and more rigid structure, they possessed higher drug loading efficiency

and it was understandable that they would release the drug more slowly and

have a relatively longer half-life. Significant increase (p<0.01) in AUC, MRT and

Vd was observed in F4 (PI, 1:25, chloroform: acetonitrile) when compared to CPT

solution and clearance was also found to be significantly low (p<0.01) when

compared with CPT in solution. Increase in MRT in plasma compared with the

same dose of CPT solution may be due to the coating of PEG on the surface of


137


polymeric micelles and sustained the release of CPT from CPT-PMs. The high

value for Vd confirms broad tissue distribution for the copolymeric micelles to the

various tissues and organs within a short period of time. In this way, the

penetration of the copolymeric micelles into tissues may be enhanced.

Hydrophilic PEG chains exposed to the aqueous surroundings may

prevent the adsorption of blood proteins onto the micelle surface and stop them

from being cleared through the RES. It was reported, that the diameter of tumour

micro vessels varies from 100 to 780 nm depending on the anatomic location of

the tumour and the tumour growth. Therefore, Me PEG-b-PCL micelles in the

size range of 100 to 500 nm would be ideal for persisting in the circulation and

accumulating in tissues with leaky vasculature such as tumours (Masato

watanabe et al., 2006).

When compared with F4 (PI, 1:25), formulation F8 (PII, 1:25) found to be

less significant (p<0.05) because of its shorter lipophilic (PCL) chain length.

These data confirm that Me-PEG-b-PCL micelles provide enhanced drug

stability in the blood and prevent the adsorption of various blood components

onto their surface. These findings also suggest that the stable incorporation of

CPT into PMs by the hydrophobic interaction of intact CPT with inner core of PMs

may be important in maintaining drug stability in the circulation.

In conclusion, the longer PCL chain length (Me PEG5000-b-PCL13, 000) led

to best long circulating properties.


138


6.10 STABILITY STUDIES

Table 39: Stability Studies of CPT Polymeric Micelles Containing Chloroform: Acetonitrile

SSttaabbiilliittyy ssttuuddiieess aarree ccaarrrriieedd oouutt aass ppeerr tthhee pprroocceedduurree eexxppllaaiinneedd iinn sseeccttiioonn 55..1188..

Formulation code

Number of days

% entrapped

drug (initial)

% entrapped drug After 15 days




RT 2-8º C RT 2-8ºC RT 2-8º C RT 2-8º C

F1 6600..33 ±± 00..88 5599..9911 ±± 00..66 5599..5511 ±± 00..55 5544..3311 ±±00..33 5588..1144 ±±00..44 5522..11 ±±00..55 56.47 ±0.8 50..51 ±0.2 56.01 ±0.8

F2 78.02 ± 0.8 7744..0011 ±± 00..99 7788..0011 ±± 00..44 7733..1111 ±±00..66 7777..6699 ±±00..99 7711..88 ±±00..33 77.15 ±0.5 68.64 ±0.6 76.52 ± 0.8

F3 8822..7755 ±± 00..77 8800..5566 ±± 00..77 8822..5599 ±± 00..55 7799..6688 ±± 00..55 8800..1199 ±± 00..66 7799..5533 ±± 00..22 80.06 ± 0.2 74.41 ± 0.9 79.61 ± 0.4

F4 8833..88 ±± 00..88 8811..99 ±± 00..55 8833..5544 ±± 00..55 8800..6633 ±± 00..22 8822..6633 ±± 00..66 79.61 ± 0.5 81.67 ± 0.4 73.64 ± 0.3 80.06 ± 0.4

F5 5566..2211 ±± 00..55 5533..1111 ±± 00..22 5566..22 ±± 00..44 5522..7711 ±± 00..66 5555..9999 ±± 00..33 5511 ±± 00..66 54.91 ± 0.8 48.77 ± 0.4 54.5 ± 0.5

F6 7777..8899 ±± 00..88 7755..2255 ±± 00..44 7766..5555 ±± 00..44 7744..1111 ±± 00..66 7766..1122 ±± 00..33 7722..8855 ±± 00..55 75.44 ± 0.6 70.61 ± 0.4 74.98 ± 0.3

F7 7799..55 ±± 00..77 7766..4444 ±± 00..55 7788..9922 ±± 00..22 7755..4488 ±± 00..55 7777..6622 ±± 00..88 74.4 ±0.9 76.91 ± 0.5 72.84 ± 0.6 76.55 ± 0.9

F8 8822 ±± 00..55 8800..0011 ±± 00..55 8822 ±± 00..55 7799..5577 ±± 00..55 8811..0066 ±± 00..55 78.88 ± 0.4 80.9 ± 0.7 74.4 ± 0.9 80 ± 0.4



139


DDaattaa IInnffeerreennccee

The CPT loaded micelle formulations did not show any noticeable change

in entrapped drug amount after 3 months when stored at 2-8°C. Around

10-13% of drug was leaked out from the polymeric micelles when they were

stored at room temperature. Lowered entrapment effeciency observed on storage

under room temperature may be due to drug expulsion from the polymeric

micelles (Otilia M.Koo et al.,). No change in colour was also observed during the

study period.

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