Formulation and optimization of temozolomide nanoparticles by 3 factor 2 level factorial design

Figures & data

Table 1. Factorial design of non-PEGylated nanoparticles bearing temozolomide

Formulation code	Independent variable level				Percent drug entrapment (%)	Particle size (nm)
	Drug (mg)	PLGA (mg)	PVA (%)	ST (sec)
1	0	-1	-1	-1	54.23	115.67
2	0	0	-1	-1	74.34	116.56
3	0	1	-1	-1	76.09	117.49
4	0	-1	0	-1	56.21	116.63
5	0	0	0	-1	77.32	117.61
6	0	1	0	-1	77.96	118.76
7	0	-1	1	-1	56.54	118.49
8	0	0	1	-1	77.43	119.49
9	0	1	1	-1	77.74	120.88
10	0	-1	-1	0	53.67	113.98
11	0	0	-1	0	73.61	115.49
12	0	1	-1	0	75.23	115.92
13	0	-1	0	0	55.61	114.96
14	0	0	0	0	76.56	116.37
15	0	1	0	0	77.21	116.84
16	0	-1	1	0	55.97	116.82
17	0	0	1	0	76.89	117.47
18	0	1	1	0	77.19	119.35
19	0	-1	-1	1	52.89	111.91
20	0	0	-1	1	72.77	113.67
21	0	1	-1	1	74.41	113.62
22	0	-1	0	1	54.32	112.83
23	0	0	0	1	75.41	113.85
24	0	1	0	1	76.1	114.98
25	0	-1	1	1	54.32	114.89
26	0	0	1	1	75.67	115.58
27	0	1	1	1	76.02	117.37
28	0	0	0	0	76.02	112.93

Table 2. Factorial design for PEGylated nanoparticles bearing temozolomide

Formulation code	Independent variable level				Percent drug entrapment (%) (PDE)	Particle size (PS) (nm)
	Drug (mg)	PLGA (mg)	PVA (%)	ST (sec)
1	0	-1	-1	-1	51.85	119.14
2	0	0	-1	-1	71.85	120.41
3	0	1	-1	-1	74.03	121.78
4	0	-1	0	-1	54.32	119.45
5	0	0	0	-1	75.01	121.2
6	0	1	0	-1	75.72	122.01
7	0	-1	1	-1	54.71	120.33
8	0	0	1	-1	75.91	122.31
9	0	1	1	-1	75.34	123.76
10	0	-1	-1	0	51.45	117.81
11	0	0	-1	0	71.25	119.32
12	0	1	-1	0	73.23	121.43
13	0	-1	0	0	53.43	118.79
14	0	0	0	0	73.83	120.32
15	0	1	0	0	75.12	121.02
16	0	-1	1	0	53.88	119.32
17	0	0	1	0	74.86	121.2
18	0	1	1	0	74.33	122.45
19	0	-1	-1	1	49.62	116.31
20	0	0	-1	1	70.37	118.32
21	0	1	-1	1	71.93	120.67
22	0	-1	0	1	52.85	113.84
23	0	0	0	1	73.12	119.21
24	0	1	0	1	73.71	119.82
25	0	-1	1	1	52.81	118.33
26	0	0	1	1	72.96	120.82
27	0	1	1	1	72.88	121.35
28	0	0	0	0	75.12	117.23

Table 3. A. Design matrix evaluation for response surface quadratic model of non-PEGylated nanoparticles bearing temozolomide

Table 3A
Degrees of freedom for evaluation
Model	14
Residuals	67
Lack of Fit	66
Pure Error	1
Corr Total	81
Table 3B
Term	Standard Error**		VIF	Ri-squared	Power at 5% α level for effect of
					0.5 Std. Dev.	1 Std. Dev.	2 Std. Dev.
A	0.136083		1	0	44.10%	95.20%	99.90%
B	0.136083		1	0	44.10%	95.20%	99.90%
C	0.136083		1	0	44.10%	95.20%	99.90%
D	0.136083		1	0	44.10%	95.20%	99.90%
AB	0.166667		1	0	31.50%	84.10%	99.90%
AC	0.166667		1	0	31.50%	84.10%	99.90%
AD	0.166667		1	0	31.50%	84.10%	99.90%
BC	0.166667		1	0	31.50%	84.10%	99.90%
BD	0.166667		1	0	31.50%	84.10%	99.90%
CD	0.166667		1	0	31.50%	84.10%	99.90%
A^2	0.233069		1.001626	0.0016234	56.10%	98.80%	99.90%
B^2	0.233069		1.001626	0.0016234	56.10%	98.80%	99.90%
C^2	0.233069		1.001626	0.0016234	56.10%	98.80%	99.90%
D^2	0.233069		1.001626	0.0016234	56.10%	98.80%	99.90%

** Basis s.d = 1.0.

Table 4. Design matrix evaluation for response surface quadratic model of PEGylated nanoparticles bearing temozolomide

Table 4A
Degrees of freedom for evaluation
Model	14
Residuals	67
Lack of Fit	66
Pure Error	1
Corr Total	81
Table 4B
Term	Standard error**	VIF	Ri-squared	Power at 5% α level for effect of
				0.5 Std. Dev.	1 Std. Dev.	2 Std. Dev.
A	0.136083	1	0	44.10%	95.20%	99.90%
B	0.136083	1	0	44.10%	95.20%	99.90%
C	0.136083	1	0	44.10%	95.20%	99.90%
D	0.136083	1	0	44.10%	95.20%	99.90%
AB	0.166667	1	0	31.50%	84.10%	99.90%
AC	0.166667	1	0	31.50%	84.10%	99.90%
AD	0.166667	1	0	31.50%	84.10%	99.90%
BC	0.166667	1	0	31.50%	84.10%	99.90%
BD	0.166667	1	0	31.50%	84.10%	99.90%
CD	0.166667	1	0	31.50%	84.10%	99.90%
A^2	0.233069	1.001626	0.0016234	56.10%	98.80%	99.90%
B^2	0.233069	1.001626	0.0016234	56.10%	98.80%	99.90%
C^2	0.233069	1.001626	0.0016234	56.10%	98.80%	99.90%
D^2	0.233069	1.001626	0.0016234	56.10%	98.80%	99.90%

Table 5. ANOVA sheet of non-PEGylated nanoparticles bearing temozolomide on percent drug entrapment

Table 5A
Response―PDE ANOVA for Response Surface Quadratic Model Analysis of variance table [Partial sum of squares-Type III]
Source	Sum of squares	df	Mean square	F-Value	p-value Prob > F
Model	10077.61	14	719.8295	81.00017	< 0.0001	significant
A-Drug	3856.411	1	3856.411	433.9499	< 0.0001
B-PLGA	3202.892	1	3202.892	360.4115	< 0.0001
C-PVA	62.46827	1	62.46827	7.02936	0.01
D-ST	43.722	1	43.722	4.919901	0.0299
AB	797.5917	1	797.5917	89.75052	< 0.0001
AC	0.831136	1	0.831136	0.093525	0.7607
AD	0.0324	1	0.0324	0.003646	0.952
BC	0.315469	1	0.315469	0.035499	0.8511
BD	0.026678	1	0.026678	0.003002	0.9565
CD	0.225625	1	0.225625	0.025389	0.8739
A^2	1116.205	1	1116.205	125.603	< 0.0001
B^2	917.3058	1	917.3058	103.2216	< 0.0001
C^2	11.23415	1	11.23415	1.264144	0.2649
D^2	2.025956	1	2.025956	0.227974	0.6346
Residual	595.4132	67	8.886765
Lack of Fit	595.2674	66	9.019204	61.86011	0.1008	not significant
Pure Error	0.1458	1	0.1458
Cor Total	10673.03	81
Table 5B
Standard deviation	2.981068	R-Squared	0.944213
Mean	63.94866	Adj R-Squared	0.932556
C.V. %	4.661658	Pred R-Squared	0.917066
PRESS	885.1574	Adeq Precision	28.43542
Table 5C
Factor	Coefficient		Standard error	95% CI low	95% CI high	VIF
	estimate	Df
Intercept	74.458	1	0.942696	72.57637	76.33963
A-Drug	8.450741	1	0.405672	7.641016	9.260465	1
B-PLGA	7.701481	1	0.405672	6.891757	8.511206	1
C-PVA	1.075556	1	0.405672	0.265831	1.88528	1
D-ST	-0.89981	1	0.405672	-1.70954	-0.09009	1
AB	4.706944	1	0.496845	3.715238	5.69865	1
AC	0.151944	1	0.496845	-0.83976	1.14365	1
AD	-0.03	1	0.496845	-1.02171	0.961706	1
BC	-0.09361	1	0.496845	-1.08532	0.898095	1
BD	0.027222	1	0.496845	-0.96448	1.018928	1
CD	-0.07917	1	0.496845	-1.07087	0.912539	1
A^2	-7.78674	1	0.694793	-9.17355	-6.39993	1.001626
B^2	-7.05896	1	0.694793	-8.44578	-5.67215	1.001626
C^2	-0.78119	1	0.694793	-2.168	0.605628	1.001626
D^2	-0.33174	1	0.694793	-1.71855	1.055073	1.001626

Final equation in terms of coded factors: PDE = 74.45+ 8.45A + 7.70B + 1.07C – 0.89D + 4.70AB + 0.15AC - 0.03AD – 0.09BC + 0.02BD – 0.07CD – 7.78A² – 7.04B² – 0.78C² – 0.33D²… Eqn. 1.

Table 6. ANOVA sheet of PEGylated nanoparticles bearing temozolomide on percent drug entrapment

Table 6A
Response―PDE | ANOVA for Response Surface Quadratic Model | Analysis of variance table [Partial sum of squares-Type III]
Source	Sum of Squares	df	Mean Square	F-Value	p-value Prob > F
							Model	9876.487	14	705.4634	75.1158	< 0.0001	Significant
A-Drug	3816.122	1	3816.122	406.3302	< 0.0001
B-PLGA	3168.176	1	3168.176	337.3387	< 0.0001
C-PVA	55.89636	1	55.89636	5.951691	0.0174
D-ST	59.09482	1	59.09482	6.292254	0.0146
AB	785.5875	1	785.5875	83.6472	< 0.0001
AC	1.1236	1	1.1236	0.119638	0.7305
AD	2.1025	1	2.1025	0.223868	0.6376
BC	8.400336	1	8.400336	0.894445	0.3477
BD	0.001736	1	0.001736	0.000185	0.9892
CD	0.298844	1	0.298844	0.03182	0.859
A^2	1082.655	1	1082.655	115.2782	< 0.0001
B^2	827.0542	1	827.0542	88.06246	< 0.0001
C^2	4.171924	1	4.171924	0.444215	0.5074
D^2	4.849833	1	4.849833	0.516397	0.4749
Residual	629.2424	67	9.391677
Lack of Fit	628.4103	66	9.521369	11.44326	0.2315	not significant
Pure Error	0.83205	1	0.83205
Cor Total	10505.73	81
Table 6B
Standard deviation	3.064584	R-Squared	0.940105
Mean	61.81098	Adj R-Squared	0.927589
C.V. %	4.957994	Pred R-Squared	0.912094
PRESS	923.5207	Adeq Precision	27.6636
Table 6C
Factor	Coefficient		Standard error	95% CI low	95% CI high	VIF
	Estimate	Df
Intercept	71.92667	1	0.969107	69.99232	73.86101
A-Drug	8.406481	1	0.417037	7.574072	9.238891	1
B-PLGA	7.65963	1	0.417037	6.82722	8.492039	1
C-PVA	1.017407	1	0.417037	0.184998	1.849817	1
D-ST	-1.04611	1	0.417037	-1.87852	-0.2137	1
AB	4.671389	1	0.510764	3.6519	5.690878	1
AC	0.176667	1	0.510764	-0.84282	1.196156	1
AD	-0.24167	1	0.510764	-1.26116	0.777823	1
BC	-0.48306	1	0.510764	-1.50254	0.536434	1
BD	0.006944	1	0.510764	-1.01254	1.026434	1
CD	-0.09111	1	0.510764	-1.1106	0.928378	1
A^2	-7.66883	1	0.714258	-9.09449	-6.24316	1.001626
B^2	-6.70272	1	0.714258	-8.12838	-5.27705	1.001626
C^2	-0.47605	1	0.714258	-1.90172	0.949617	1.001626
D^2	-0.51327	1	0.714258	-1.93894	0.912394	1.001626

Final Equation in terms of Coded factors: PDE = 71.92+ 8.40A + 7.65B + 1.01C – 1.04D + 4.67AB + 0.17AC - 0.24AD – 0.48BC + 0.01BD – 0.09CD – 7.66A² – 6.70B² – 0.47C² – 0.51D²…. Eqn. 3.

Table 7. ANOVA sheet of non-PEGylated nanoparticles bearing temozolomide on particle size

Table 7A
Response―PS ANOVA for Response Surface Quadratic Model Analysis of variance table [Partial sum of squares-Type III]
Source	Sum of squares	df	Mean square	F-value	p-value Prob > F
Model	1086.671	14	77.61934	287.9431	< 0.0001	Significant
A-Drug	715.4784	1	715.4784	2654.198	< 0.0001
B-PLGA	51.58756	1	51.58756	191.3735	< 0.0001
C-PVA	113.9414	1	113.9414	422.6863	< 0.0001
D-ST	171.842	1	171.842	637.4793	< 0.0001
AB	0.103469	1	0.103469	0.383839	0.5377
AC	0.297025	1	0.297025	1.101868	0.2976
AD	0.028336	1	0.028336	0.105118	0.7468
BC	0.238469	1	0.238469	0.884646	0.3503
BD	0.005625	1	0.005625	0.020867	0.8856
CD	0.092011	1	0.092011	0.341332	0.561
A^2	28.44803	1	28.44803	105.5332	< 0.0001
B^2	0.016815	1	0.016815	0.062377	0.8035
C^2	4.484401	1	4.484401	16.6357	0.0001
D^2	0.506602	1	0.506602	1.879333	0.175
Residual	18.06084	67	0.269565
Lack of Fit	12.14404	66	0.184001	0.031098	1	not significant
Pure Error	5.9168	1	5.9168
Cor Total	1104.732	81
Table 7B
Standard deviation	0.519196	R-Squared	0.983651
Mean	115.273	Adj R-Squared	0.980235
C.V. %	0.450406	Pred R-Squared	0.977501
PRESS	24.85564	Adeq Precision	70.73664
Table 7C
Factor	Coefficient		Standard error	95% CI low	95% CI high	VIF
	estimate	Df
Intercept	115.856	1	0.164184	115.5283	116.1837
A-Drug	3.64	1	0.070654	3.498975	3.781025	1
B-PLGA	0.977407	1	0.070654	0.836382	1.118433	1
C-PVA	1.452593	1	0.070654	1.311567	1.593618	1
D-ST	-1.78389	1	0.070654	-1.92491	-1.64286	1
AB	-0.05361	1	0.086533	-0.22633	0.119109	1
AC	0.090833	1	0.086533	-0.08189	0.263553	1
AD	0.028056	1	0.086533	-0.14466	0.200776	1
BC	0.081389	1	0.086533	-0.09133	0.254109	1
BD	-0.0125	1	0.086533	-0.18522	0.16022	1
CD	0.050556	1	0.086533	-0.12216	0.223276	1
A^2	-1.24311	1	0.121008	-1.48464	-1.00158	1.001626
B^2	0.030222	1	0.121008	-0.21131	0.271756	1.001626
C^2	0.493556	1	0.121008	0.252022	0.735089	1.001626
D^2	-0.16589	1	0.121008	-0.40742	0.075645	1.001626

Final Equation in terms of coded factors: PS = 115.85+ 3.64A + 0.97B + 1.45C – 1.78D – 0.05AB + 0.09AC + 0.03AD – 0.08BC – 0.01BD – 0.05CD – 1.24A² – 0.03B² – 0.49C² – 0.16D²…. Eqn. 2.

Table 8. ANOVA sheet for PEGylated nanoparticles bearing temozolomide on particle size

Table 8A
Source	Sum of squares	df	Mean square	F-value	p-value Prob > F
Model	749.4104	14	53.52932	128.02	< 0.0001	Significant
A-Drug	471.6476	1	471.6476	1127.986	< 0.0001
B-PLGA	101.9563	1	101.9563	243.8373	< 0.0001
C-PVA	67.15645	1	67.15645	160.6104	< 0.0001
D-ST	57.57736	1	57.57736	137.7012	< 0.0001
AB	0.005625	1	0.005625	0.013453	0.908
AC	0.1444	1	0.1444	0.345345	0.5587
AD	0.001344	1	0.001344	0.003215	0.9549
BC	0.056803	1	0.056803	0.135849	0.7136
BD	0.595469	1	0.595469	1.424116	0.2369
CD	0.0441	1	0.0441	0.105469	0.7464
A^2	42.29364	1	42.29364	101.1489	< 0.0001
B^2	0.43066	1	0.43066	1.029961	0.3138
C^2	8.163949	1	8.163949	19.52479	< 0.0001
D^2	0.00791	1	0.00791	0.018917	0.891
Residual	28.01488	67	0.418133
Lack of Fit	23.24083	66	0.352134	0.07376	0.9995	not significant
Pure Error	4.77405	1	4.77405
Cor Total	777.4253	81
Table 8B
Standard deviation	0.646632	R-Squared	0.963965
Mean	118.9363	Adj R-Squared	0.956435
C.V. %	0.543679	Pred R-Squared	0.949605
PRESS	39.1785	Adeq Precision	46.84067
Table 8C
Factor	Coefficient		Standard error	95% CI low	95% CI high	VIF
	Estimate	Df
Intercept	119.6103	1	0.204483	119.2022	120.0185
A-Drug	2.95537	1	0.087995	2.779731	3.13101	1
B-PLGA	1.374074	1	0.087995	1.198434	1.549714	1
C-PVA	1.115185	1	0.087995	0.939546	1.290825	1
D-ST	-1.03259	1	0.087995	-1.20823	-0.85695	1
AB	0.0125	1	0.107772	-0.20261	0.227614	1
AC	-0.06333	1	0.107772	-0.27845	0.15178	1
AD	0.006111	1	0.107772	-0.209	0.221225	1
BC	0.039722	1	0.107772	-0.17539	0.254836	1
BD	0.128611	1	0.107772	-0.0865	0.343725	1
CD	-0.035	1	0.107772	-0.25011	0.180114	1
A^2	-1.51573	1	0.15071	-1.81655	-1.21491	1.001626
B^2	-0.15295	1	0.15071	-0.45377	0.147867	1.001626
C^2	0.665938	1	0.15071	0.365121	0.966756	1.001626
D^2	-0.02073	1	0.15071	-0.32155	0.280089	1.001626

Final equation in terms of coded factors: PS = 119.61+ 2.95A + 1.37B + 1.11C – 1.03D + 0.01AB - 0.06AC + 0.01AD + 0.03BC + 0.02BD – 0.03CD – 1.51A² – 0.01B² + 0.66C² – 0.02D²…. Eqn. 4.

Figure 1. Graph showing the effect of drug and PLGA, drug and PVA, drug and ST, PLGA and PVA, PLGA and ST and PVA and ST on percent drug entrapment of non-PEGylated nanoparticles bearing temozolomide.

Figure 2. Graph showing effect of drug and PLGA, drug and PVA, drug and ST, PLGA and PVA, PLGA and ST and PVA and ST on particle size of non-PEGylated nanoparticles bearing temozolomide.

Figure 3. Graph showing effect of drug and PLGA, drug and PVA, drug and ST, PLGA and PVA, PLGA and ST and PVA and ST on percent drug entrapment of PEGylated nanoparticles bearing temozolomide.

Figure 4. Graph showing effect of drug and PLGA, drug and PVA, drug and ST, PLGA and PVA, PLGA and ST and PVA and ST on particle size of PEGylated nanoparticles bearing temozolomide.

Figure 5. Scanning electron microscopy of PEGylated nanoparticles.

Table 9. Variables and their levels in polynomial quadratic model

	Low (-1)	Medium (0)	High (+1)
A- Drug Concentration	2.5 mg	5.0 mg	7.5 mg
B- PLGA Concentration	25 mg	50 mg	75 mg
C- PVA Concentration	0.50%	1.00%	1.50%
D- Sonication Time	30 Sec	60 Sec	90 Sec
Dependent variables
Y1- Percent Drug Entrapment (PDE)
Y2- Particle Size (PS)

Dillen K, Vandervoort J, Mooter GV. Verheyden, Ludwig A. Factorial design, physicochemical characterization and activity of ciprofloxacin-PLGA nanoparticles. Int J Pharm 2004; 275:171 - 87; http://dx.doi.org/10.1016/j.ijpharm.2004.01.033; PMID: 15081148

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