OPTIMIZATION, FORMULATION AND CHARACTERIZATION OF …shodhganga.inflibnet.ac.in/bitstream/10603/18214/12/12_chapter 05.… · batches. The effect of formulation variables on characteristics

CHAPTER - 5

OPTIMIZATION, FORMULATION AND

CHARACTERIZATION OF MICROSPHERES

5.1 Optimization of Method 5.2 Preparation of Microspheres

5.3 Characterization of Microspheres 5.4 Result & Discussion

OPTIMIZATION, FORMULATION & CHARACTERIZATION 113

5.1 OPTIMIZATION OF METHOD

5.1.1 Process optimization

It is the discipline of adjusting a process so as to optimize some specified set of

parameters without violating some constraint. The most common goals are minimizing

cost, maximizing throughput, and/or efficiency. This is one of the major quantitative

tools in industrial decision making. Traditional optimization methods generally study the

effect of one variable at a time, because it is statistically easier to manipulate. However,

in many cases, two factors may be interdependent, and it is impractical or false to

attempt to analyze them in the traditional way. A 32 randomized full factorial design was

adopted to optimize the variables.

5.1.2 The three-level design

The three-level design is written as a 3k factorial design. It means that k factors are

considered, each at 3 levels. These are (usually) referred to as low, intermediate and high

levels. These levels are numerically expressed as 0, 1, and 2. One could have considered

the digits -1, 0, and +1. The reason that the three-level designs were proposed is to model

possible curvature in the response function and to handle the case of nominal factors at 3

levels. A third level for a continuous factor facilitates investigation of a quadratic

relationship between the response and each of the factors.

In the present investigation two factors were evaluated, each at 3 levels (low, medium

and high), and experimental trials were performed at all nine possible combinations. In

the study, temperature (X1) and stirring speed (X2) were selected as independent

variables. The particle size, % drug entrapment, and % Buoyancy were selected as

dependent variables(80).


5.1.3 Statistical Analysis

Statistical analysis of factorial design batches was performed by multiple regression

analysis using Microsoft Excel. To evaluate the contribution of each factor with different

levels on responses, two way analysis of variance (ANOVA) followed by Tukey test was

performed using Sigma Stat 2.03 (SPSS, Chicago, IL). To demonstrate graphically the

influence of each factor (stirring speed and temperature) on dependent variables,

response surface plot were generated, using Sigma Plot Software Version 8.0, (Jindal

Scientific Software, San Rafeal, CA). The probability level p<0.05 was considered to be

significant(81).

5.2 PREPARATION OF MICROSPHERES

Microspheres were prepared by emulsification extraction technique. Pectin was used as a

polymer and casein as emulsifier. The microspheres were prepared by modification of

method described by Bulgareli et al.

In preliminary batches 10 ml of 15% w/v (in different ratio) casein and pectin

solution were added to 60ml Soya oil. Both oil and polymer solution was preheated

separately up to 60˚C. Each drug was added to the polymer emulsifier solution in two

different quantities (50mg and 100mg) as shown in table 5.1 (a, b, and c). The mixture

was mechanically stirred at 1000rpm to form o/w emulsion, after 5 min the solution was

rapidly cooled at 15°C. 250ml of acetone was added to dehydrate & flocculate

coacervate droplets. The microspheres was isolated by filtration through sintered glass

filter. Residual oil over the microspheres was removed by washing with 250ml of

acetone. After preparation of microspheres they were stored at room temperature in a

dessicator at 8% relative humidity, otherwise drying conditions can influence

microsphere release profile. Fifty millimeter diameter vessel, a three blade turbine rotator

of 35 mm in diameter with digital stirring speed counter was selected(125, 126 and 127). In

factorial design batches M1 to M9, D1 to D9 and F1 to F9, the polymer to emulsifier


ratio (1:1) and quantity of drug (100mg) was kept constant which was selected from

preliminary batches. The temperature and stirring speed were varied in batches as shown

in table 5.3 (a, b and c). All other variables were used as mentioned in preliminary trial

batches. The effect of formulation variables on characteristics of the microspheres is

summarized in Table 5.1 and 5.3 and in Figure 5.1 (a, b, and c), 5.2 (a, b, and c) and 5.3

(a, b, and c).

Table 5.1(a): Results of preliminary trial batches of Methotrexate loaded Microsphere.

Batch Code

Polymer to Emulsifier Ratio (mg)

Drug Quantity (mg)

Drug Entrapment (%)

Bouyancy (%)

Particle Size and Sphericity

MP1 1250:250 50 95.32±0.89 50.0±0.79 Small MP2 1000:500 50 94.29±0.57 59.0±0.63 Small MP3 750:750 50 94.01±1.34 74.0±0.95 Small/Spherical/Free

flowing MP4 500:1000 50 78.94±2.01 75.0±0.88 Large/Irregular MP5 250:1250 50 72.38±0.99 76.0±0.92 Large/Irregular MP6 1250:250 100 97.75±1.24 59.0±0.80 Small MP7 1000:500 100 96.26±0.73 67.0±0.69 Small MP8 750:750 100 96.10±1.10 83.0±1.22 Small/Spherical/Free

flowing MP9 500:1000 100 82.01±0.83 84.0±0.96 Large MP10 250:1250 100 75.33±1.39 86.0±0.87 Large/Irregular * n = 3, all values ± standard deviation, statistically significant at 0.05 level

Table 5.1(b): Results of preliminary trial batches of Doxorubicin Loaded Microspheres Batch Code


Drug Quantity (mg)

Drug Entrapment (%)

Bouyancy (%)


DP1 1250:250 50 74.07±1.05 60.0±0.88 Small DP2 1000:500 50 73.53±0.94 64.0±0.63 Small DP3 750:750 50 72.95±0.87 82.0±0.77 Small/Spherical/Free

flowing DP4 500:1000 50 57.65±1.19 84.0±1.38 Large/Irregular DP5 250:1250 50 50.74±0.66 86.0±1.29 Large/Irregular DP6 1250:250 100 76.44±0.84 61.0±0.95 Small DP7 1000:500 100 75.17±0.90 63.0±0.76 Small DP8 750:750 100 74.23±1.01 84.0±0.84 Small/Spherical/Free

flowing DP9 500:1000 100 58.24±0.98 85.0±0.94 Large DP10 250:1250 100 54.32±0.85 88.0±0.60 Large/Irregular * n = 3, all values ± standard deviation, statistically significant at 0.05 level


Table 5.1(c): Results of preliminary trial batches of 5-Fluorouracil Loaded Microspheres Batch Code


Drug Quantity (mg)

Drug Entrapment (%)

Bouyancy (%)


FP1 1250:250 50 75.98±0.83 58.0±0.86 Small FP2 1000:500 50 74.01±0.98 62.0±0.93 Small FP3 750:750 50 73.35±0.88 76.0±1.05 Small/Spherical/Free

flowing FP4 500:1000 50 59.99±0.68 77.0±0.99 Large/Irregular FP5 250:1250 50 54.23±0.49 78.0±1.04 Large/Irregular FP6 1250:250 100 77.83±0.84 60.0±0.95 Small FP7 1000:500 100 75.39±0.97 61.0±0.67 Small FP8 750:750 100 74.02±0.92 77.0±0.72 Small/Spherical/Free

flowing FP9 500:1000 100 61.66±0.63 79.0±1.21 Large/Irregular FP10 250:1250 100 58.74±0.59 80.0±1.37 Large/Irregular * n = 3, all values ± standard deviation, statistically significant at 0.05 level

5.3 CHARACTERIZATION OF MICROSPHERES

5.3.1 Quantitative Analysis of Microspheres

The drug content of microsphers was determined by milling & immersing the

microsphere of each drug, in distilled water after which they were stirred for 4 hrs & left

at room temperature overnight. Filtered by using Whatmann filter paper, volume was

made up by washing the residue & assayed in UV spectrophotometer. The absorbance

was determined at λmax of respective drug against blank. The quantity of methotrexate,

doxorubicin and 5-FU microencapsulated was calculated from standard calibration curve

of drugs(61,62,64). The results are recorded in table 5.2(a, b, and c).

Table 5.2(a): Drug Content Of Methotrexate Loaded Microsphere

S. No Amount of Drug incorporated (mg)

Amount of drug recovered (mg)

% Yield

M1 100 95.40±0.45 83.21 M2 100 95.45±0.34 83.43 M3 100 95.89±0.37 84.00 M4 100 96.68±0.63 82.43 M5 100 96.64±0.50 82.19 M6 100 96.66±0.59 84.54 M7 100 96.96±0.30 83.65 M8 100 97.16±0.78 83.02 M9 100 97.54±0.48 84.21

* n = 3, all values ± standard deviation, statistically significant at 0.05 level


Table 5.2(b): Drug Content Of Doxorubicin Loaded Microsphere


Amount of Drug recovered (mg)

% Yield

D1 100 78.4±0.31 75.00 D2 100 78.9±0.80 73.12 D3 100 79.2±0.41 73.22 D4 100 78.1±0.42 75.35 D5 100 78.3±0.52 74.77 D6 100 79.2±0.53 75.68 D7 100 78.2±0.63 75.42 D8 100 78.6±0.10 75.76 D9 100 79.0±0.25 74.42 * n = 3, all values ± standard deviation, statistically significant at 0.05 level

Table 5.2(c): Drug Content Of 5-Fluorouracil Loaded Microsphere


Amount of Drug recovered (mg)

% Yield

F1 100 90.24±0.65 83.65 F2 100 92.41±0.44 84.65 F3 100 93.48±0.39 83.21 F4 100 92.41±0.42 84.32 F5 100 93.41±0.74 84.19 F6 100 94.51±0.42 85.32 F7 100 92.52±0.84 84.42 F8 100 93.89±0.52 85.30 F9 100 94.90±0.34 85.62 * n = 3, all values ± standard deviation, statistically significant at 0.05 level

5.3.2 Determination Of Drug Entrapment Efficiency And Percentage Yield Microspheres 200mg were crushed in a dry glass pestle mortar, mixed with distilled

water, and then filtered with 0.2m membrane filter and aliquot of the filtrate was diluted

with distilled water. The filtrate was analyzed for drug content and absorbance was

determined at λmax of respective drug by using blank The drug entrapment efficiency was

calculated by the following formula(68, 69, 70). The results of preliminary and factorial

batches are recorded in table 5.1(a, b, and c), and 5.3(a, b, and c) respectively for

methotrexate, doxorubicin and 5-flourouracil microspheres.

Percentage drug entrapment: {Practical drug content/ Theoretical drug content}*100

Percentage Yield: {Weight of microspheres /Weight of polymer and Drug}*100


50

60

70

80

90

100

110

-1.0

-0.5

0.0

0.5

1.0

-1.0

-0.5

0.0

0.5

PA

RTIC

LE S

IZE (M

ICRO

METER)

TEM

PERATU

RE

(C)

STIRRING SPEED (RPM)

Fig 5.1(a): Response Surface Plot Of Methotraxate Microsphere For Particle Size

72

74

76

78

80

82

84

-1.0

-0.5

0.0

0.5

1.0

-1.0

-0.5

0.0

0.5

% B

uoya

ncy

Tempe

rature (C

)

Stirring Speed (rpm)

Fig 5.1(b): Response Surface Plot Of Methotraxate Microsphere % Buoyancy.


95.0

95.5

96.0

96.5

97.0

97.5

98.0

-1.0

-0.5

0.0

0.5

1.0

-1.0

-0.5

0.0

0.5

% D

rug

Ent

rapm

ent

Tempe

rature (C

)


Fig 5.1(c): Response Surface Plot Of Methotraxate Microsphere % Drug

Entrapment

50

60

70

80

90

100

-1.0

-0.5

0.0

0.5

1.0

-1.0

-0.5

0.0

0.5

Particle S

ize

Tempe

rature(C

)

Stirring speed(rpm)

Fig 5.2(a): Response Surface Plot Of Doxorubicin Microsphere For Particle Size


72.5

73.0

73.5

74.0

74.5

75.0

75.5

76.0

76.5

-1.0

-0.5

0.0

0.5

1.0

-1.0

-0.5

0.0

0.5

% D

rug

Ent

rapm

ent

Tem

pera

ture

(C)


Fig 5.2(b): Response Surface Plot Of Doxorubicin Microsphere

72

74

76

78

80

82

84

-1.0

-0.5

0.0

0.5

1.0

-1.0

-0.5

0.0

0.5

% B

uoya

ncy

Tem

perature (C

)


Fig 5.2(c): Response Surface Plot Of Doxorubicin Microsphere For % Bouyancy


.

40

50

60

70

80

90

100

110

-1.0

-0.5

0.0

0.5

1.0

-1.0

-0.5

0.0

0.5

Particle Size

Tem

perature (C

)


Fig No. 5.3(a) Response Surface Plot Of 5-FU Microsphere

90

91

92

93

94

95

96

-1.0

-0.5

0.0

0.5

1.0

-1.0

-0.5

0.0

0.5

% D

rug

Ent

rapm

ent

Tempe

ratu

re (C

)


Fig 5.3(b): Response Surface Plot Of 5-FU Microsphere


75

76

77

78

79

80

81

82

83

-1.0

-0.5

0.0

0.5

1.0

-1.0

-0.5

0.0

0.5

% B

ouya

ncy

Tem

perature (C

)


Fig 5.3(c): Response Surface Plot Of 5-FUMicrosphere

Table 5.3(a): Formulation Characteristics Of Batches In A 32 Full Factorial Design* For Methotrexate Loaded Microspheres.

Batch Code

Coded Value Particle size (µm)

Buoyancy (%)

Drug Entrapment (%)

X1 X2

M1 -1 -1 107.00±0.23 74.2±0.23 95.40±0.89

M2 -1 0 86.00±0.45 78.0±0.34 95.45±0.48

M3 -1 +1 76.66±0.35 80.3±0.66 95.89±0.67

M4 0 -1 98.30±0.26 75.2±0.34 96.68±0.29

M5 0 0 79.40±0.39 78.4±0.25 96.64±0.88

M6 0 +1 64.00±0.98 81.0±0.65 96.66±0.64

M7 +1 -1 88.00±0.23 76.0±0.37 96.69±0.97

M8 +1 0 74.80±0.48 79.5±0.42 97.16±0.48

M9 +1 +1 59.60±0.95 82.0±0.27 97.54±0.53

Coded Values

Actual Values

Variable levels

X1 X2

–1 40 500 Low

0 50 1000 Medium

1 60 1500 High

* n = 3, all values ± standard deviation, statistically significant at 0.05 level. X1 is temperature of both phases (ºC), and X2 is stirring speed (rpm). All batches contained 200 mg methotrexate, 1.5% pectin and casein in ratio 1:1.


Table 5.3(b): Formulation Characteristics Of Batches In A 32 Full Factorial Design* For Doxorubicin Loaded Microspheres.

Batch Code


Buoyancy (%)

Drug Entrapment (%)

X1 X2

D1 -1 -1 94.2±0.66 73.2±0.47 72.97±0.54

D2 -1 0 89.0±0.49 78.0±0.87 73.65±0.49

D3 -1 +1 80.0±0.95 80.0±0.55 74.37±0.40

D4 0 -1 70.0±0.64 74.2±0.63 73.97±0.58

D5 0 0 62.2±0.87 79.0±0.86 75.41±0.74

D6 0 +1 55.0±0.44 82.0±0.29 75.99±0.38

D7 +1 -1 77.5±0.85 75.0±0.86 73.89±0.99

D8 +1 0 70.3±0.64 79.9±0.65 75.01±0.28

D9 +1 +1 59.2±0.53 81.0±0.88 75.86±0.65

Coded Values

Actual Values

Variable levels

X1 X2

–1 40 500 Low

0 50 1000 Medium

1 60 1500 High



Table 5.3(c): Formulation Characteristics Of Batches In A 32 Full Factorial Design* For 5-Fluorouracil Loaded Microsphere.

Batch Code


Buoyancy (%)

Drug Entrapment (%)

X1 X2

F1 -1 -1 102.0±0.57 38.0±0.63 72.4±0.59

F2 -1 0 75.0±0.34 48.0±0.52 72.2±0.87

F3 -1 +1 58.0±0.75 42.0±0.96 72.2±0.66

F4 0 -1 96.0±0.65 69.0±0.83 72.5±0.49

F5 0 0 69.0±0.88 70.0±0.88 72.5±0.55

F6 0 +1 54.0±0.39 78.0±0.76 72.4±0.37

F7 +1 -1 90.0±0.92 73.0±0.59 73.3±0.82

F8 +1 0 61.0±0.45 75.0±0.44 73.2±0.54

F9 +1 +1 50.0±0.86 76.0±0.36 73.3±0.74

Coded Values

Actual Values

Variable levels

X1 X2

–1 40 500 Low

0 50 1000 Medium

1 60 1500 High



5.3.3 Determination Of Particle Size And Size Distribution The particle size of the microsphere was determined by using optical microscopy

method. Approximately 500 particles were counted for particle size using a calibrated

optical microscope. The particle size of preliminary batches and factorial batches for

individual drug is reported in table 5.1 (a, b, and c) and 5.3 (a, b, and c), respectively for

methotrexate, doxorubicin and 5-flourouracil microspheres (10, 11).

5.3.4 Morphological Study Of Microspheres

The shape and surface morphology of the microsphere was investigated using scanning

electron microscopy as shown in figure 5.4 a, b, c, d, e and f for methotrexate,

doxorubicin, 5-FU microspheres, surface of microspheres, and microspheres in group,

respectively. Photomicrographs were taken at 50x magnification operated with an

acceleration voltage of 10kV and working distance 9.1mm was maintained(89,92).

5.4(a): Methotrexate Microsphere 5.4(b): Doxorubicin Microsphere

5.4(c): 5-FU Microsphere 5.4(d): Surface Of Microsphere


Fig.5.4(e): Scanning Electron Photomicrograph Of Microspheres

Fig.5.4(f): Scanning Electron Photomicrograph Of Microspheres


5.3.5 Percentage Buoyancy Porous microspheres 200mg were spread over the surface of a USP XXIV paddle type

dissolution apparatus filled with 900ml of buffer containing 0.02% v/v tween 20. The

mixture was stirred at 100rpm. Particles were pipetted out and separated by filtration.

Particles in sinking particulate layer were again separated by filtration. Particles of both

type were dried in dessicator until constant weight was obtained. Both fractions of the

microsphere were weighed and percentage buoyancy was determined by using following

formula and the results are recorded in table 5.1 (a, b, and c), and 5.3 (a, b, and c),

respectively for methotrexate, doxorubicin and 5-flourouracil microspheres(92).

% Buoyancy = {[wf / wf + ws] x 100}

Where, wf = weight of floating microspheres, ws = weight of sinking microspheres

5.3.6 Stability Of Microsphere At Different Gastric pH

Gastro-retentive floating microspheres are low-density systems that have sufficient

buoyancy to float over gastric contents and remain in stomach for prolonged period

where they are exposed to different pH and different enzymatic conditions which can

influence their physicochemical properties and drug release behavior and can alter their

stability characteristics. To test this hypothesis, drug loaded microspheres were subjected

to different pH media where they encountered different ionic strengths and enzymatic

conditions and the change in their properties was elucidated by counter checking their

particle size. pH dependent stability studies were carried out in following media:

1. pH 1.1: 12 ml HCl (32%) with 1188 ml H2O

2. pH 3.5: 150 ml solution (10.5 g citric acid+100 ml NaOH (1 M)+395.5 ml H2O)

with 100 ml HCl

3. Simulated Gastric Fluid (SGF): 0.2% NaCl, Pepsin 0.7% HCl with pH 1.2


Ten milliliters of simulated fluid were added to 10 mg of microspheres. The samples

were analyzed after a period of 8hrs in each of the above media. The above time intervals

were selected for the study based on expected formulation residence time in stomach.

Particle size was determined on the preset time periods(83). The results are recorded in

table 5.4.

Table 5.4: Initial And Final Particle Size After Exposure To Different Gastric pH. Medium Initial Size (µm) Final size (µm)

MM DM FM MM DM FM pH 1.1 59.60±0.95 59.21±0.37 50.0±0.28 60.20±0.46 61.22±0.19 51.01±0.88

pH 3.5 59.60±0.95 59.21±0.37 50.0±0.28 61.30±0.82 60.42±0.49 51.35±0.53 SGF 59.60±0.95 59.21±0.37 50.0±0.28 60.99±0.29 61.02±0.36 52.09±0.41

MM- Methotrexate Microsphere, DM- Doxorubicin Microsphere, and FM- 5Flourouracil Microsphere, * n = 3, all values ± standard deviation, statistically significant at 0.05 level 5.3.7 Fourier Transforms Infrared Spectroscopy Drug polymer interaction was studied by FT-IR spectroscopy (Shimadzu Affinity I, FT-

IR spectrophotometer). The spectrum was recorded for pure drugs, loaded drug

microspheres and unloaded microsphere (placebo). Samples were prepared by mixing

5% of drug or microsphere with 95% of KBr in glass pestle mortar. The scanning range

was 4000 cm-1 to 400 cm-1 and resolution was 2 cm-1(79). The spectra are recorded in

figure 5.5(a, b, c and d).

5.3.8 Flow Properties

Flow properties were determined in terms of Carr’s index (Ic) and Hausner’s ratio (HR).

The Carr index is an indication of the compressibility of a powder. The Carr index is

frequently used in pharmaceutics as an indication of the flowability of a powder. A

Carr’s index greater than 25 is considered to be an indication of poor flowability, and

below 15, of good flowability

Ic = ρt - ρb / ρt

Where, ρt = tapped density ρb = bulk density


400500600700800900100012001400160018002000240028003200360040001/cm

6

8

10

12

14

16

18

20

22

24

26

28

30

32

34

%T

2927.9

4 2856.5

8

1737.8

6

1624.0

6

1442.7

5

1230.5

8

1145.7

2

1099.4

3

1022.2

7

954.7

6

894.9

7

833.2

5

Fig 5.5(a): FTIR Spectra Of Plecebo Microsphere

400500600700800900100012001400160018002000240028003200360040001/cm

4.5

6

7.5

9

10.5

12

13.5

15

16.5

18

19.5

21

22.5

24

25.5

%T

29

29

.87

28

56

.58

17

37

.86

14

42

.75

114

5.7

2

10

24

.20

95

2.8

4

83

1.3

2

Fig 5.5(b): FTIR Spectra Of Methotrexate Microsphere


400500600700800900100012001400160018002000240028003200360040001/cm

6

8

10

12

14

16

18

20

22

24

26

28

30

32

34

36

%T

29

27

.94

28

56

.58

17

34

.01

16

20

.21

14

42

.75

11

45

.72

10

22

.27

95

6.6

9

Fig 5.5(c): FTIR Spectra Of Doxorubicin Microsphere

400500600700800900100012001400160018002000240028003200360040001/cm

6

7.5

9

10.5

12

13.5

15

16.5

18

19.5

21

22.5

24

25.5

27

28.5

30

31.5

33%T

29

31

.80

28

56

.58

17

35

.93

16

41

.42

14

42.7

5

11

45.7

2

10

99

.43

102

4.2

0

95

4.7

6

83

1.3

2

Fig 5.5(d): FTIR Spectra of 5FU Microsphere.


Table 5.5: Angle Of Repose, Carr’s Index And Hausner’s Ratio As An Indication Of Flow Properties.

Angle of repose (θ) Carr’s index (%) Hausner’s ratio Type of flow >20 5-15 - Excellent

20-30 12-16 <1.25 Good 30-40 18-21 - Fair to passable

- 25-35 >1.25 Poor - 33-38 1.25-1.5 Very poor

>40 >40 - Extremely poor

Table 5.6(a): Micromeritic Properties Of Methotrexate Microsphere.


Table 5.6(b): Micromeritic Properties Of Doxorubicin Microspheres.

Code Angle of Repose (ө) Carr’s Index (%) Hausner’s Ratio

D1 29.43±0.432 15.13±0.343 1.166±0.042 D2 27.34±0.322 14.98±0.274 1.159±0.025 D3 25.94±0.426 13.67±0.294 1.143±0.026 D4 26.62±0.078 16.46±0.331 1.158±0.047 D5 23.23±0.632 12.49±0.532 1.149±0.038 D6 22.67±0.325 11.63±0.362 1.139±0.054 D7 26.89±0.522 14.55±0.380 1.160±0.035 D8 25.45±0.376 12.23±0.265 1.154±0.022 D9 23.23±0.534 11.89±0.121 1.147±0.048


Table 5.6(c): Micromeritic Properties Of 5-FU Microspheres.


F1 27.03±0.543 16.42±0.562 1.173±0.025 F2 25.27±0.420 15.18±0.376 1.142±0.034 F3 22.63±0.350 13.34±0.598 1.131±0.047 F4 25.92±0.244 16.66±0.548 1.159±0.032 F5 23.13±0.532 13.29±0.431 1.134±0.056 F6 21.67±0.222 12.11±0.643 1.130±0.049 F7 24.54±0.320 15.25±0.254 1.159±0.065 F8 22.45±0.534 14.24±0.436 1.142±0.032 F9 20.23±0.867 11.23±0.543 1.132±0.045



M1 27.36±0.690 17.17±0.241 1.164±0.015 M2 24.67±0.508 15.14±0.362 1.131±0.021 M3 22.64±0.321 12.45±0.423 1.123±0.040 M4 25.64±0.423 16.46±0.352 1.132±0.028 M5 22.32±0.034 12.78±0.423 1.125±0.019 M6 20.53±0.540 11.03±0.242 1.120±0.042 M7 24.34±0.231 14.95±0.632 1.142±0.024 M8 23.95±0.321 12.67±0.531 1.140±0.033 M9 20.32±0.432 11.01±0.342 1.135±0.035


The Carr index is related to the Hausner ratio, another indication of flowability, by the

formula:

HR = ρt / ρb The Hausner ratio and Carr’s index are both measures of the flow properties of

powders. A Hausner ratio of <1.25 indicates a powder that is free flowing

whereas >1.25 indicates poor flow ability. The smaller the Carr’s Index the better the

flow properties. For example 5-15 indicates excellent, 12-16 good, 18-21 fair and > 23

poor flow(128).

The angle of repose (θ) of the microsphere, which measures the resistance to particle

flow, was determined by the fixed funnel method, using the following equation:

tan θ = 2H/D

Where, 2H/D is the surface area of the free standing height of the heap that formed after

making the microspheres flow from the glass funnel(129).

When bulk granular materials are poured onto a horizontal surface, a conical pile

will form. The internal angle between the surface of the pile and the horizontal surface is

known as the angle of repose and is related to the density, surface area and shapes of the

particles, and the coefficient of friction of the material. Material with a low angle of

repose forms flatter piles than material with a high angle of repose.

Fixed Funnel Method The material is poured through a funnel to form a cone. The tip of

the funnel should be held close to the growing cone and slowly raised as the pile grows,

to minimize the impact of falling particles. Stop pouring the material when the pile

reaches a predetermined height or the base a predetermined width. Rather than attempt to

measure the angle of the resulting cone directly, divide the height by half the width of the

base of the cone. The inverse tangent of this ratio is the angle of repose. The results are

recorded in table 5.6.


5.4 RESULT AND DISCUSSION

Porous microspheres of drugs were successfully prepared by emulsification extraction

method. A statistical model incorporating interactive polynomial term was used to

evaluate the response

Y = b0 + b1X1 + b2X2 + b12X1X2 + b11X11 + b22X22

Where, Y is the dependent variable, b0 is the arithmetic mean response of nine runs, b1 is

the estimated coefficient for the factor X1. The main effects (X1 and X2) represent the

average results of changing one factor at a time from its low to high value. The

interaction terms (X1X2) show how the responses change when two factors are

simultaneously changed. The polynomial terms (X1X1 and X2X2) are included to

investigate nonlinearity. The fitted equation relating the responses particle size, % drug

entrapment, and % buoyancy to the transformed factor are shown in equation 1, 2, 3, 4,

5, 6, 7, 8, and 9 for methotrexate, doxorubicin HCl and 5-FU microspheres respectively.

PS = 1.06 x12 - 0.09 x1 x2 + 1.81 x2

2 - 8.39 x1 - 16.02 x2 + 79.36 Eq-1

%DE = -0.26 x12 + 0.0225 x1 x2 + 0.105 x2

2 + 0.82 x1 + 0.175 x2 + 96.59 Eq-2

%B = -0.2 x12 + 0.15 x1 x2 + 0.05 x2

2 + 1.3 x1 + 2.75 x2 + 78 Eq-3

PS = 15.96 x12 - 1.02 x1 x2 - 1.183 x2

2 - 9.36 x1 - 7.91 x2 + 63.18 Eq-4

%DE = -0.83 x12 + 0.14 x1 x2 – 0.18 x2

2 + 0.62 x1 + 0.89 x2 + 75.24 Eq-5

%B = -0.55 x12 - 0.2 x1 x2 - 1.4 x2

2 + 0.78 x1 + 3.43 x2 + 79.33 Eq-6

PS = -0.331 x12 + 1 x1 x2 + 6.66 x2

2 - 5.66 x1 - 21 x2 + 68.55 Eq-7

%DE = -0.56 x12 - 0.215 x1 x2 - 0.25 x2

2 + 0.863 x1 + 1.28 x2 + 93.63 Eq-8

%B = 1.15 x12 + 0.125 x1 x2 + 0.45 x2

2 + 1.15 x1 + 1.81 x2 + 77.13 Eq-9

To demonstrate graphically the effect of the temperature and stirring speed, the response

surface plots Figure 5.1(a, b, c), 5.2(a, b, c), and 5.3(a, b, c) were generated for the


dependent variables, particle size, % drug entrapment and %buoyancy using Sigma Plot

software.

Results of ANOVA for the measured responses are provided in Table 5.10, 5.11 and

5.12. The statistical analysis of the factorial design batches was performed by multiple

polynomial regression analysis using Microsoft Excel. The data clearly depicts that the

Particle size (PS), % drug entrapment (%DE), and % Buoyancy (%B) values are strongly

dependent on the selected independent variables. The polynomial equations can be used

to draw conclusions after considering the magnitude of coefficient and the mathematical

sign it carries (positive or negative). The value of the correlation coefficient indicates a

good fit (Table 5.7, 5.8 and 5.9).

Table 5.7: Multiple Regression Output for Dependent Variables* (methotrexate microsphere).

Parameters Coefficient of Regression Parameters B0 b1 b2 B12 b11 b22 r P PS 79.36 -8.39 -16.02 -0.09 1.06 1.81 0.991 <0.001 %EE 78.00 1.3 2.75 0.15 -0.2 0.05 0.991 <0.001 %B 96.59 0.82 0.175 0.022 -0.26 0.10 0.969 <0.001 PS, particle size; %EE, % drug entrapment; %B, % buoyancy.

Table 5.8: Multiple Regression Output for Dependent Variables*(doxorubicin microsphere)

Parameters Coefficient of Regression Parameters B0 b1 b2 b12 b11 b22 r P PS 68.55 -5.66 -21 1.0 0.33 6.66 0.983 <0.001 %EE 77.13 1.15 1.81 0.125 1.15 0.45 0.964 <0.001 %B 93.58 0.78 1.37 -0.34 -0.48 -0.17 0.960 <0.001 PS, particle size; %EE, % drug entrapment; %B, % buoyancy.

Table 5.9: Multiple Regression Output for Dependent Variables*(5-flourouracil microspheres)

Parameters Coefficient of Regression Parameters B0 b1 b2 b12 b11 B22 r P PS 68.55 -5.66 -21 1.0 0.33 6.66 0.983 <0.001 %EE 77.13 1.15 1.81 0.125 1.15 0.45 0.964 <0.001 %B 93.58 0.78 1.37 -0.34 -0.48 -0.17 0.960 <0.001 PS, particle size; %EE, % drug entrapment; %B, % buoyancy.


Table 5.10: Results of Analysis of Variance for Measured Response* (methotrexate microspheres).

Parameters df SS MS F Significance F For PS

Regression 2 19063.172 981.586 163.363 <0.001 Residual 6 36.052 6.009 Total 8 1999.223 249.903

For %EE Regression 2 4.218 2.019 46.652 <0.001 Residual 6 0.271 0.0452 Total 8 4.489 0.561

For %B Regression 2 55.515 27.757 159.373 <0.001 Residual 6 1.045 0.174 Total 8 56.560 7.070 *df indicates degree of freedom; SS, sum of square; MS, mean sum of square; and F, Fischer’s ratio. Table 5.11: Results of Analysis of Variance for Measured Response* (doxorubicin HCl microspheres).




For %B Regression 2 76.142 38.071 58.000 <0.001 Residual 6 3.938 0.656 Total 8 80.080 10.010 *df indicates degree of freedom; SS, sum of square; MS, mean sum of square; and F, Fischer’s ratio.


Table 5.12: Results of Analysis of Variance for Measured Response* (5-FU microspheres).




For %B Regression 2 27.737 13.868 25.343 <0.001 Residual 6 3.283 0.547 Total 8 31.020 3.878 *df indicates degree of freedom; SS, sum of square; MS, mean sum of square; and F, Fischer’s ratio.

To evaluate the contribution of different levels of factor (X1) and factor (X2), 2-way

ANOVA followed by Tukey test was performed using Sigma Stat software. Results of

two-way ANOVA for the both factors at different levels are provided in Table 5.13, 5.14

and 5.15. Where p is the parameter used when computing q. The larger the p, the larger q

needs to be to indicate a significant difference. p is an indication of the differences in the

ranks of the group means being compared. Groups means are ranked in order from

largest to smallest, and p is the number of means spanned in the comparison. For

example, when comparing four means, comparing the largest to the smallest p = 4, and

when comparing the second smallest to the smallest p = 2.

If a group is found to be not significantly different than another group, all groups with p

ranks in between the p ranks of the two groups that are not different are also assumed not

to be significantly different, and a result of DNT (Do Not Test) appears for those

comparisons. The Difference of Means is a gauge of the size of the difference between

the groups or cells being compared.


Table 5.13: Results of two way ANOVA for factors X1 and X2 at different levels (methotrexate microspheres).

Particle size – Factor X1 Comparison Diff of Means p q P P<0.050 -1.000 vs. 1.000 16.787 3 11.139 0.003 Yes -1.000 vs. 0.000 9.453 3 6.273 0.025 Yes 0.000 vs. 1.000 7.333 3 4.866 0.056 No

Factor X2 -1.000 vs. 1.000 32.047 3 21.265 <0.001 Yes -1.000 vs. 0.000 17.833 3 11.834 0.003 Yes 0.000 vs. 1.000 14.213 3 9.432 0.006 Yes

%Drug entrapment – Factor X1 1.000 vs. -1.000 1.640 3 16.826 <0.001 Yes 1.000 vs. 0.000 0.560 3 5.745 0.033 Yes 0.000 vs. -1.000 1.080 3 11.081 0.003 Yes

Factor X2

1.000 vs. -1.000 0.350 3 3.591 0.131 No 1.000 vs. 0.000 0.280 3 2.873 0.220 Do Not Test 0.000 vs. -1.000 0.0700 3 0.718 0.872 Do Not Test

% Bouyancy – Factor X1 1.000 vs. -1.000 2.600 3 9.192 0.007 Yes 1.000 vs. 0.000 1.100 3 3.889 0.106 No 0.000 vs. -1.000 1.500 3 5.303 0.043 Yes

Factor X2 1.000 vs. -1.000 5.500 3 19.445 <0.001 Yes 1.000 vs. 0.000 2.800 3 9.899 0.005 Yes 0.000 vs. -1.000 2.700 3 9.546 0.006 Yes

X1 is temperature of both phases (ºC), and X2 is stirring speed (rpm) p is a parameter used when computing q. The larger the p, the larger q needs to be to indicate a significant difference. p is an indication of the differences in the ranks of the group means being compared. Groups means are ranked in order from largest to smallest, and p is the number of means spanned in the comparison.


Table 5.14: Results of two way ANOVA for factors X1 and X2 at different levels (doxorubicin HCl microspheres).

Particle size – Factor X1

Comparison Diff of Mean p q P P<0.050 -1.000 vs. 1.000 5.733 3 4.824 0.057 No -1.000 vs. 0.000 5.400 3 4.543 0.069 Do Not Test 0.000 vs. 1.000 0.333 3 0.280 0.979 Do Not Test

Factor X2

-1.000 vs. 1.000 27.733 3 23.334 <0.001 Yes -1.000 vs. 0.000 5.400 3 4.543 0.069 No 0.000 vs. 1.000 22.333 3 18.791 <0.001 Yes

% Drug entrapment – Factor X1

0.000 vs. -1.000 1.460 3 11.655 0.003 Yes 0.000 vs. 1.000 0.203 3 1.623 0.539 No 1.000 vs. -1.000 1.257 3 10.032 0.005 Yes

Factor X2

1.000 vs. -1.000 1.797 3 14.342 0.001 Yes 1.000 vs. 0.000 0.717 3 5.721 0.034 Yes 0.000 vs. -1.000 1.080 3 8.621 0.008 Yes

% Bouyancy – Factor X1

1.000 vs. -1.000 1.900 3 57.000 <0.001 Yes 1.000 vs. 0.000 0.900 3 27.000 <0.001 Yes 0.000 vs. -1.000 1.000 3 30.000 <0.001 Yes

Factor X2

1.000 vs. -1.000 6.867 3 206.000 <0.001 Yes 1.000 vs. 0.000 2.033 3 61.000 <0.001 Yes 0.000 vs. -1.000 4.833 3 145.000 <0.001 Yes

X1 is temperature of both phases (ºC), and X2 is stirring speed (rpm).


Table 5.15: Results of two way ANOVA for factors X1 and X2 at different levels (5-Fluorouracil microspheres).

Particle size – Factor X1 Comparison Diff of Mean p q P P<0.050 -1.000 vs. 1.000 11.333 3 12.555 0.002 Yes -1.000 vs. 0.000 5.333 3 5.908 0.030 Yes 0.000 vs. 1.000 6.000 3 6.647 0.020 Yes

Factor X2 -1.000 vs. 1.000 42.000 3 46.529 <0.001 Yes -1.000 vs. 0.000 27.667 3 30.650 <0.001 Yes 0.000 vs. 1.000 14.333 3 15.879 <0.001 Yes

% Drug entrapment – Factor X1 1.000 vs. -1.000 1.727 3 8.932 0.007 Yes 1.000 vs. 0.000 0.300 3 1.552 0.565 No 0.000 vs. -1.000 1.427 3 7.380 0.014 Yes

Factor X2

1.000 vs. -1.000 2.573 3 13.312 0.002 Yes 1.000 vs. 0.000 1.033 3 5.345 0.042 Yes 0.000 vs. -1.000 1.540 3 7.966 0.011 Yes

% Bouyancy – Factor X1

1.000 vs. 0.000 2.300 3 16.494 <0.001 Yes 1.000 vs. -1.000 2.300 3 16.494 <0.001 Yes -1.000 vs. 0.000 0.000 3 0.000 1.000 No

Factor X2 1.000 vs. -1.000 3.633 3 26.056 <0.001 Yes 1.000 vs. 0.000 2.267 3 16.255 <0.001 Yes 0.000 vs. -1.000 1.367 3 9.801 0.005 Yes

X1 is temperature of both phases (ºC), and X2 is stirring speed (rpm).


The porous microspheres of pectin were prepared by emulsification extraction technique.

Pectin was selected as polymer for the preparation of porous microspheres owing to its

biodegradable nature and stability at lower pH properties. In preliminary batches

different ratio of polymer to emulsifier was used for preparing the polymer solution, the

polymer solution was too viscous at ratio 1250:250 and 1000: 500 (pectin: casein) and

difficult to pour in oil. As well as quantity of polymer increased from 250-1250mg in

polymer to emulsifier ratio, percentage entrapment efficiency of microspheres increased

with low % buoyancy. On the other hand if quantity of emulsifier was raised 250 to

1250 in polymer to emulsifier ratio, microspheres produced were irregular and of large

size with increased buoyancy and with less entrapment efficiency. Therefore 750:750

(1:1) ratio of pectin to casein was found to be optimum concentration of polymer and

emulsifier which provide microspheres of small size with good % entrapment efficiency

and increased % buoyancy.

Two different quantities of drug 50mg and 100mg were used for preparation of

microspheres. 100mg of drug can be easily incorporated in microspheres, but no

significant effect of amount of drug on % entrapment efficiency and on % buoyancy

were seen (table 5.1 a, b and c).

On the basis of preliminary trials 32 full factorial design was employed to study

the effect of independent variables temperature and stirring speed on dependent variables

particle size, percentage buoyancy and percentage drug entrapment efficiency. The

results were depicted in Table 5.3 (a, b, and c) and in Figure 5.1(a, b, and c), 5.2 (a, b,

and c), and 5.3 (a, b, and c).

Microspheres were characterized for drug content. Drug content ranges from

95.40±0.45 to 97.54±0.48, 78.1±0.42 to 79.2±0.53 and 90.24±0.65 to 94.90±0.34,

respectively for methotrexate microsphere, doxorubicin microsphere and 5-FU

microspheres. The maximum % drug entrapment was found to be 95.40±0.89 to


97.54±0.53, 72.97±0.54 to 75.99±0.38 and 72.2±0.87 to 73.3±0.74 methotrexate

microsphere, doxorubicin microsphere and fluorouracil microsphere, respectively. The

results are presented in table 5.2 (a, b, and c). This proves that emulsification extraction

technique is a proper method of preparation of porous microspheres and that the

polymers and oil have been rightly selected.

Particle size and surface morphology were assessed by scanning electron

microscopy. Photomicrographs showed that microspheres are spherical with rough

surface. It was observed that the particle size of methotrexate microspheres, doxorubicin

microspheres and 5- fluorouracil microspheres were 107.00±0.23 to 59.60±0.95,

94.2±0.66 to 55.0±0.44 and 102.0±0.57 to 50.0±0.86 micrometer, respectively. The

results were depicted in table 5.3 (a, b, and c). Size of microspheres greatly affects the

flow properties. Particles or microspheres having a smaller size showed good flow

properties as shown in Figure 5.4 (a, b, c and d).

The % Buoyancy was found to be 74.2±0.23 to 82.0±0.27, 73.2±0.47 to

82.0±0.29 and 38.0±0.63 to 76.0±0.36 for methotrexate microspheres, doxorubicin

microspheres and 5-fluorouracil microspheres, respectively. The results were depicted in

Table 5.3 (a, b, and c).

The GI stability of the particles was investigated by suspending the particles to

simulated GI fluids and found to be quite stable (Table 5.4) under the study conditions

and duration. This formed an important exercise, as stable particles would remain floated

and result in increase in subsequent bioavailability of drug.

Figure 5.5 (a, b, c and d) are the characteristic peaks of the plecebo microspheres

and drug loaded microspheres. Drug polymer interaction was studied by FT-IR

spectroscopy. The spectrum was recorded for pure drugs, loaded drug microspheres and

unloaded microspheres (placebo). Samples were prepared by mixing 5% of drug or

microspheres with 95% of KBr in glass pestle mortar. The scanning range was 4000 cm-1


to 400 cm-1 and resolution was 2 cm-1. The peaks which are present in spectra of placebo

microspheres are similar to that of drug loaded microspheres. FTIR analysis reveals that

complete encapsulation of drug occurs in microspheres.

Flow properties of the formulations were determined and it is found that angle of

Repose, Housner’s Ratio and Car’s Index, for methotrexate microsphere was

20.32±0.432 to 27.36±0.690, 11.01±0.342 to 17.17±0.241, and 1.135±0.035 to

1.164±0.015, respectively. For doxorubicin microsphere it was 22.67±0.325 to

29.43±0.432, 11.63±0.362 to 15.13±0.343, and 1.139±0.054 to 1.166±0.042

respectively. For 5- fluorouracil microsphere it was 20.23±0.867 to 27.03±0.543,

11.23±0.543 to 16.42±0.562 and 1.132±0.045 to 1.173±0.025, respectively. Angle of

repose is defined as the miximum angle possible between the surface of a pile of the

powder and the horizontal plane. The lower the angle of repose, better the flow property.

The rough and irregular surface of particles gives higher angle of repose. The Hausner

ratio and Carr’s index are both measures of the flow properties of powders. A Hausner

ratio of <1.25 indicates a powder that is free flowing whereas >1.25 indicates poor flow

ability. The smaller the Carr’s Index the better the flow properties. For example 5-15

indicates excellent, 12-16 good, 18-21 fair and > 23 poor flow. So for the optimized

formulation angle of repose is low, Hausner ratio of <1.25 and smaller Carr’s Index

which means the formulations are free flowing. The results are shown in table 5.6.

From the results of Tukey test, it was found that both factor X1 and X2 had

significant effect on particle size, % buoyancy and % drug entrapment at different levels

as shown in table 5.13, 5.14 and 5.15.

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