Table 1 Formulation composition of Capecitabine loaded PLGA based nanoparticles fabricated as per BBD.
Sl No
|
Batch Code
|
Conc. of PLGA(mg)
|
Conc. of Poloxamer 188(%)
|
Amplitude of Sonication(%)
|
Particle Size(nm)
|
%EE
|
PDI
|
X1
|
X2
|
X3
|
Y1
|
Y2
|
Y3
|
1
|
NP1
|
0.00
|
-1.00
|
1.00
|
218.6±5.5
|
65.08±5.25
|
0.662±0.022
|
2
|
NP2
|
-1.00
|
1.00
|
0.00
|
198.5±3.5
|
58.09±3.51
|
0.711±0.012
|
3
|
NP3
|
1.00
|
0.00
|
-1.00
|
230.04±6.5
|
70.55±2.25
|
0.255±0.015
|
4
|
NP4
|
-1.00
|
0.00
|
-1.00
|
199.95±3.2
|
59.01±3.55
|
0.69±0.005
|
5
|
NP5
|
1.00
|
0.00
|
1.00
|
219.55±5.8
|
71.55±4.55
|
0.184±0.023
|
6
|
NP6
|
1.00
|
1.00
|
0.00
|
220.05±4.9
|
70.08±3.65
|
0.195±0.013
|
7
|
NP7
|
1.00
|
-1.00
|
0.00
|
219.85±5.0
|
72.01±4.26
|
0.17±0.019
|
8
|
NP8
|
0.00
|
-1.00
|
-1.00
|
219.01±5.5
|
66.98±3.15
|
0.573±0.025
|
9
|
NP9
|
0.00
|
0.00
|
0.00
|
218.45±5.2
|
65.02±4.05
|
0.515±0.014
|
10
|
NP10
|
-1.00
|
-1.00
|
0.00
|
194.75±6.0
|
57.98±3.09
|
0.915±0.025
|
11
|
NP11
|
0.00
|
1.00
|
1.00
|
217.5±5.8
|
59.01±3.35
|
0.88±0.015
|
12
|
NP12
|
0.00
|
0.00
|
0.00
|
218.09±3.9
|
58.11±4.15
|
0.538±0.032
|
13
|
NP13
|
0.00
0.00
|
0.00
|
0.00
|
234.27±4.6
|
56.95±2.95
|
0.592±0.018
|
14
|
NP14
|
0.00
|
0.00
|
217.98±5.9
|
57.75±2.99
|
0.851±0.021
|
15
|
NP15
|
0.00
|
0.00
|
0.00
|
219.99±4.0
|
58.22±3.26
|
0.577±0.023
|
16
|
NP16
|
-1.00
|
0.00
|
1.00
|
200.05±4.5
|
59.09±3.28
|
0.566±0.013
|
17
|
NP17
|
0.00
|
1.00
|
-1.00
|
218.02±3.5
|
59.08±2.27
|
0.161±0.011
|
All Observed results were presented as mean ± standard deviation, where n = 3
Levels
X1(mg) 30 40 50
X2(%) 0.5 1.00 1.5
X3(%) 40 50 60
Table 2 Quality Target Product Profile (QTPP) for nanoformulations
QTPP
|
Target
|
Justification
|
Dosage form
|
Nanoformulations
|
To improve stability and bioavailability
|
Route of administration
|
Oral
|
Self medication, non-invasive
|
Physical form
|
Lyophilized powder
|
Elegant appearance and stable
|
Physicochemical characterization
|
Entrapment Efficiency
Particle size
Zetapotential
PDI
Drug release
|
To ensure drug loading
To enhance bioavailability
To ensure stability
Uniform dispersibility
Influence pharmacokinetics of drug
|
API solubility in carrier system
|
High up to 50%
|
Influence on drug release pattern along with therapeutic effect
|
Stability
|
Quality requirement
|
Influence on quality of the product
|
Container and closer system
|
Appropriate for the dosage form
|
Ensuring target shelf life
|
Pharmacokinetics
|
Absorption
Distribution
Metabolism
Targeting
|
Required for desired efficacy of the drug.
|
Table 3 List of most commonly Critical Quality Attributes (CQAs) for Nanoformulations
CQAs
|
Target
|
Is this a CQA
|
Justicatoions
|
Partcle size
|
< 250 nm
|
Yes
|
Small size of particles ensure more absorption and different cancer cell targeting
|
Zetapotential
|
>±25 mV
|
Yes
|
Ensures stability of globules
|
PDI
|
< 1
|
Yes
|
Ensures uniform dispersibility
|
Entrapment Efficiency
|
As high as possible
|
Yes
|
Required for desired dose delivery, sustain release, reduce volume of administration and maximize therapeutic efficacy.
|
Drug Release
|
Prolong release(>12h)
|
Yes
|
Slow and predetermined release to attain prolonged drug absorption
|
Table 4 Critical Material attributes and risk assessment for nanoformulations
|
Critical Material Attributes
|
Drug product CQAs
|
Drug
|
Polymer(PLGA)
|
Stabilizer concentration(Poloxamer188)
|
Particle size
|
Low
|
Medium
|
High
|
PDI
|
Low
|
Medium
|
High
|
Zeta potential
|
Low
|
Medium
|
High
|
Entrapment Efficiency
|
High
|
High
|
Medium
|
Drug Release
|
Medium
|
High
|
Low
|
Table 5 Justification for the initial risk assessment of the material attributes
Drug Product CQAs
|
Critical Material Attributes
|
Justifications
|
Size/PDI
|
Drug
Polymers
Stabilizer
|
Drug has no effect on size of particles as it is dispersed in polymeric solutions.
The size of particles may vary with concentration of polymers
The size of particles decreases with increase in concentrations of stabilizer as it stabilizes the nanoformulations and prevents aggregations.
|
Entrapment Efficiency
|
Drug
Polymers
Stabilizer
|
The physicochemical properties of the drug may influence the entrapment efficiency. The more the lipophilicity of the drug the more is the chances of encapsulation.
The optimum level of polymers required for maximizing encapsulation.
The stabilizer has little effect on encapsulation.
|
Drug Release
|
Drug
Polymers
Stabilizer
|
The properties of drug have medium effect on drug release.
The polymers have much influence on drug release as more is the concentration of the polymers less is the drug release.
The stabilizer has no effect on drug release.
|
Zeta potential
|
Drug
Polymers
Stabilizer
|
The drug has little effect on zeta potential
The charge of polymers influences zeta potential up to certain extent.
The stabilizer has crucial role on zeta potential as it imparts charge.
|
Table 6 Justification of the initial risk assessment for manufacturing process
Critical Process Attributes
|
Drug Product CQAs
|
Justification
|
Probe sonication amplitude
|
Size/PDI
Entrapment Efficiency
Drug Release
Zetapotential
|
Size is influenced by the amplitude of probe sonication
Size reduction may lead to poor drug encapsulation due to leaching in case of hydrophilic drugs
Size reduction has no influence on drug release
Zeta potential is seldom affected by size reduction
|
Size reduction time
|
Size/PDI
Entrapment Efficiency
Drug Release
Zetapotential
|
Sonication time highly influences the particle size
Entrapment efficiency seldom affected by sonication time
Drug release seldom affected by sonication time
Zeta potential seldom seldom affected by sonication time
|
Stirring speed
|
Size/PDI
Entrapment Efficiency
Drug Release
Zeta potential
|
The more is the stirring speed the lesser is the particle size and less stirring speed leads to agglomeration.
Entrapment efficiency is not influenced by the stirring speed.
The drug release is not influenced by stirring speed
The Zeta potential is seldom affected by stirring speed.
|
Table 7 Summary of Failure Mode Effective Analysis (FMEA) showing RPN scores for various formulation and process parameters influencing the CQAs.
Sl No
|
Failure mode
|
S(Severity)
|
O(Occurrence)
|
D(detection)
|
RPN(SOD)
|
Failure effect
|
1
|
PLGA conc.
|
7
|
6
|
6
|
252
|
PS, EE, PDI,ZP
|
2
|
PLGA:Eudragit S100
|
5
|
6
|
4
|
120
|
PS, EE
|
3
|
Stirring time
|
7
|
5
|
5
|
175
|
EE, PS
|
4
|
Stirring speed
|
6
|
3
|
4
|
72
|
EE, PS
|
5
|
Sonication time
|
5
|
6
|
3
|
90
|
PS, PDI, ZP
|
6
|
Sonication amplitude
|
8
|
5
|
5
|
200
|
PS, PDI
|
7
|
Conc. of Poloxamer 188
|
8
|
6
|
5
|
240
|
PS, PDI, EE
|
Table 8 Formulation and process variables along with their high and low levels scrutinized utilizing Taguchi design.
Runs
|
PLGA conc.
|
PLGA:Eudragit S100
|
Stirring time
|
Stirring speed
|
Sonication time
|
Sonication amplitude
|
Conc. of Poloxamer 188
|
1
|
-1
|
+1
|
+1
|
+1
|
+1
|
-1
|
-1
|
2
|
+1
|
+1
|
-1
|
-1
|
+1
|
+1
|
-1
|
3
|
+1
|
-1
|
+1
|
-1
|
+1
|
-1
|
+1
|
4
|
+1
|
+1
|
-1
|
+1
|
-1
|
-1
|
+1
|
5
|
-1
|
-1
|
-1
|
+1
|
+1
|
+1
|
+1
|
6
|
-1
|
-1
|
-1
|
-1
|
-1
|
-1
|
-1
|
7
|
-1
|
+1
|
+1
|
-1
|
-1
|
+1
|
+1
|
8
|
+1
|
-1
|
+1
|
+1
|
-1
|
+1
|
-1
|
Factors under investigation
|
Levels
|
|
|
Low High
|
|
PLGA Conc.
|
|
PLGA:Eudragit S100
PLGA:Eudragit S100
Stirring speed
Stirring time
Sonication time
Sonication amplitude
Conc. of Poloxamer 188
|
30 50
1:1 1:5
300rpm 1500rpm
0.5h 2h
2s 5s
30% 90%
0.5% 1.5%
|
Table 9 In vitro cytotoxicity assay of Capecitabine and Capecitabine loaded PLGA based nanoparticles against HT29 cell lines
Concentration(mcg/mL)
|
% Inhibition
|
Capecitabine
|
Optimized PLGA based Nanoparticles
|
0.001
|
3.68
|
8.97
|
0.01
|
7.84
|
12.84
|
0.1
|
31.97
|
36.49
|
1
|
45.37
|
48.42
|
10
|
51.64
|
73.16
|
IC50 value(mcg/mL)
|
2
|
1
|
Table 10 Pharmacokinetic parameters of Capecitabine and Capecitabine nanosuspension after oral administration
Pharmacokinetic parameters
|
Pure drug(Capecitabine)
|
Capecitabine loaded Nanosuspension
|
Cmax (mcg/mL)
|
0.0583±0.004
|
0.0898±0.005
|
Tmax (h)
|
6±1.59
|
4.5±1.41
|
AUC 0-∞ (mcg.h/mL)
|
0.4247±0.06
|
1.1367±0.08
|
MRT 0-∞ (h)
|
10.39±1.75
|
11.27±1.76
|
Ke(1/h)
|
3.336817±0.12
|
6.457382±0.22
|
T1/2(h)
|
0.207683±0.03
|
0.107319±0.04
|
Vd(mL)
|
398.1988±10.55
|
29.10717±1.95
|
Cl (mL/h)
|
97.12529±2.56
|
187.9561±4.59
|
Note: Data conferred as Mean±SEM, n=3. Cmax, Peak plasma concentration; Tmax, time to achieve peak plasma concentration; Ke, elimination rate constant; Vd, Volume of distribution; AUC 0-∞ : area under the curve from time of administration to infinite time; Cl: clearance, Vd: Volume of distribution, T1/2: Elimination half life.