Process optimization for the preparation of oligomycin-loaded folate-conjugated chitosan nanoparticles as a tumor-targeted drug delivery system using a two-level factorial design method

Figures & data

Figure 1 Schematic description of the preparation procedure of FA-Oli-CSNPs.

Abbreviations: Oli-A, oligomycin-A; FA-CS, the folate-conjugated chitosan; TPP, sodium tripolyphosphate; FA-Oli-CSNPs, folate-conjugated chitosan nanoparticles loading oligomycin-A; NHS-folate, N-Hydroxysuccinimide ester of folate.

Table 1 Factors and their levels in 2-LFD analysis

Independent variables	Symbol	Coded levels
		−1	0	1
FA-CS concentration (mg/mL)	X1	0.5	4.25	8
Oli-A concentration (mg/mL)	X2	0.5	5.25	10
Mass ratio of FA-CS to TPP	X3	0.5	5.25	10
TPP concentration (mg/mL)	X4	0.5	1.75	3
Crosslinking time (h)	X5	0.5	2.25	4

Abbreviations: FA-CS, folate-conjugated chitosan; Oli-A, oligomycin-A; TPP, sodium tripolyphosphate; 2-LFD, two-level factorial design.

Table 2 Experimental design and results of the 2-LFD analysis

Run	Variables					Responses
	X1	X2	X3	X4	X5	MPS (nm)	DER (%)	DLR (%)	ZP (mV)
1	8	0.5	10	0.5	4	435.6	8.4	0.5	22.2
2	4.25	5.25	5.25	1.75	2.25	298.3	27.8	25.5	28.7
3	8	10	10	3	4	478.1	8.5	9.6	46.2
4	0.5	10	0.5	0.5	0.5	419.1	6.6	56.5	20.1
5	0.5	10	10	3	0.5	82.1	5.3	51.4	20.3
6	0.5	0.5	0.5	3	0.5	700.4	5.5	5.2	14.0
7	8	0.5	0.5	0.5	0.5	184.4	23.0	1.4	21.5
8	8	10	0.5	3	0.5	74.1	8.0	9.0	25.6
9	8	0.5	0.5	3	4	185.7	30.2	1.9	35.2
10	4.25	5.25	5.25	1.75	2.25	251.7	24.9	23.6	26.9
11	4.25	5.25	5.25	1.75	2.25	275.0	26.4	24.5	27.8
12	8	10	10	0.5	0.5	513.2	3.4	4.0	33.7
13	8	0.5	10	3	0.5	993.1	5.6	0.4	47.2
14	0.5	0.5	10	3	4	126.1	1.6	1.6	21.7
15	0.5	10	0.5	3	4	150.2	6.2	55.2	26.8
16	0.5	0.5	10	0.5	0.5	301.0	2.6	2.6	20.9
17	0.5	10	10	0.5	4	526.0	34.4	87.3	18.4
18	8	10	0.5	0.5	4	241.3	0.5	0.7	28.4
19	0.5	0.5	0.5	0.5	4	502.5	16.5	14.2	14.1

Notes: FA-CS concentration (X₁), Oli-A concentration (X₂), mass ratio of FA-CS to TPP (X₃), TPP concentration (X₄), and crosslinking time (X₅).

Abbreviations: MPS, mean particle size; DER, drug entrapment rate; DLR, drug loading rate; ZP, zeta potential; FA-CS, folate-conjugated chitosan; Oli-A, oligomycin-A; 2-LFD, two-level factorial design.

Figure 2 The FTIR spectroscopy figure of NHS-FA compared with FA.

Notes: NHS-FA presents two remarkable absorption peaks at 1660 cm⁻¹ due to the N–O bond, and at 1700 cm⁻¹ because of the C=O bond. This phenomenon proves that FA has been successfully transferred into NHS-FA.

Abbreviations: FA, Folate; NHS-FA, N-hydroxysuccinimide ester of folate; NHS, N-hydroxysuccinimide.

Figure 3 Determination of NHS-FA conjugation with CS.

Notes: (a) CS has an extremely weak absorption peak at 300 nm. (b) FA-CS has three absorption peaks, at 281 nm, 300 nm, and 343 nm, respectively. (c) NHS-FA has a strong absorption peak at 281 nm and a weak absorption peak at 343 nm.

Abbreviations: CS, chitosan; FA-CS, the folate-conjugated chitosan; NHS-FA, N-hydroxysuccinimide ester of folate.

Table 3 Estimated effects and coefficients for MPS and DLR (coded units)

Term	MPS					DLR
	Net effect	Regression coefficient	Standard error	Standardized effect (t)	P	Net effect	Regression coefficient	Standard error	Standardized effect (t)	P
Constant		369.55	5.819	63.51	0.0000		18.84	0.2488	75.75	0.0000
X1	37.27	18.64	5.819	3.20	0.0852	−30.82	−15.41	0.2488	−61.94	0.0003
X2	−118.10	−59.05	5.819	−10.15	0.0096	30.76	15.38	0.2488	61.84	0.0003
X3	124.70	62.35	5.819	10.72	0.0086	1.67	0.83	0.2488	3.35	0.0787
X4	−41.66	−20.83	5.819	−3.58	0.0699	−4.12	−2.06	0.2488	−8.27	0.0143
X5	−77.75	−38.88	5.819	−6.68	0.0217	5.05	2.53	0.2488	10.15	0.0096
X1 × X2	−4.92	−2.46	5.819	−0.42	0.7134	−25.97	−12.99	0.2488	−52.21	0.0004
X1 × X3	308.94	154.47	5.819	26.55	0.0014	−1.27	−0.64	0.2488	−2.56	0.1245
X1 × X4	130.78	65.39	5.819	11.24	0.0078	7.68	3.84	0.2488	15.43	0.0042
X1 × X5	−28.31	−14.15	5.819	−2.43	0.1355	−5.59	−2.79	0.2488	−11.23	0.0078
X2 × X3	53.99	27.00	5.819	4.64	0.0435	6.08	3.04	0.2488	12.22	0.0066
X2 × X4	−187.11	−93.56	5.819	−16.08	0.0038	−1.70	−0.85	0.2488	−3.14	0.0764
X2 × X5	154.52	77.26	5.819	13.28	0.0056	2.89	1.45	0.2488	5.82	0.0283
X3 × X4	17.56	8.78	5.819	1.51	0.2703	−3.75	−1.87	0.2488	−7.53	0.0172
X3 × X5	−3.20	−1.60	5.819	−0.27	0.8094	5.12	2.56	0.2488	10.30	0.0093
X4 × X5	−149.64	−74.82	5.819	−12.86	0.0060	−4.49	−2.25	0.2488	−9.03	0.0121

Notes: FA-CS concentration (X₁), Oli-A concentration (X₂), mass ratio of FA-CS to TPP (X₃), TPP concentration (X₄), and crosslinking time (X₅).

Abbreviations: MPS, mean particle size; DLR, drug loading rate.

Figure 4 Pareto chart of the standardized effects for (A) MPS and (B) DLR – full model.

Abbreviations: MPS, mean particle size; DLR, drug loading rate.

Table 4 Analysis of variance for MPS and DLR using adjusted SS for tests

Source	MPS					DLR
	Degrees of freedom	Sum of squares (SS)	Mean square (MS)	F	P-value	Degrees of freedom	Sum of squares (SS)	Mean square (MS)	F	P-value
X1	1	5556.58	5556.58	10.26	0.0852	1	3798.87	3798.87	3837.12	0.0003
X2	1	55793.98	55793.98	102.99	0.0096	1	3785.94	3785.94	3824.05	0.0003
X3	1	62196.62	62196.62	114.81	0.0086	1	11.12	11.12	11.23	0.0787
X4	1	6942.64	6942.64	12.82	0.0699	1	67.73	67.73	68.41	0.0143
X5	1	24181.03	24181.03	44.64	0.0217	1	102.01	102.01	103.04	0.0096
X1 × X2	1	96.97	96.97	0.18	0.7134	1	2698.28	2698.28	2725.45	0.0004
X1 × X3	1	3.818E + 005	3.818E + 005	704.72	0.0014	1	6.50	6.50	6.57	0.1245
X1 × X4	1	68417.56	68417.56	126.30	0.0078	1	235.78	235.78	238.15	0.0042
X1 × X5	1	3204.98	3204.98	5.92	0.1355	1	124.88	124.88	126.14	0.0078
X2 × X3	1	11660.22	11660.22	21.52	0.0435	1	147.74	147.74	149.23	0.0066
X2 × X4	1	1.400E + 005	1.400E + 005	258.51	0.0038	1	11.49	11.49	11.61	0.0764
X2 × X5	1	95504.18	95504.18	176.30	0.0056	1	33.52	33.52	33.86	0.0283
X3 × X4	1	1233.94	1233.94	2.28	0.2703	1	56.18	56.18	56.74	0.0172
X3 × X5	1	40.86	40.86	0.075	0.8094	1	104.96	104.96	106.02	0.0093
X4 × X5	1	89567.02	89567.02	165.34	0.0060	1	80.64	80.64	81.45	0.0121
Error	2	1083.45	541.73			2	1.98	0.99
Total	17	94728.03				17	11267.62

Notes: MPS: S = 23.28 R² = 99.89%; R² _(adj) = 99.03%; F = adj · MS_factor/adj · MS_Error DLR: S = 1.00 R² = 99.98%; R² _(adj) = 99.85%; F = adj · MS_factor/adj · MS_Error.

Table 5 Estimated effects and coefficients for MPS and DLR – reduced model

Term	MPS				Term	DLR
	Net effect	Regression coefficient	Standard error	P		Net effect	Regression coefficient	Standard error	P
Constant		369.55	14.39	0.0000	Constant		18.84	2.10	0.0000
X2	−118.10	−59.05	14.39	0.0027	X1	−30.82	−15.41	2.10	0.0000
X3	124.70	62.35	14.39	0.0019	X2	30.76	15.38	2.10	0.0000
X5	−77.75	−38.88	14.39	0.0243	X1 × X2	−25.97	−12.99	2.10	0.0000
X1 × X3	308.94	154.47	14.39	0.0000
X1 × X4	130.78	65.39	14.39	0.0014
X2 × X4	−187.11	−93.56	14.39	0.0001
X2 × X5	154.52	77.26	14.39	0.0005
X4 × X5	−149.64	−74.82	14.39	0.0006

Abbreviations: MPS, mean particle size; DLR, drug loading rate.

Figure 5 Pareto chart of the standardized effects for (A) MPS and (B) DLR – reduced model.

Abbreviations: MPS, mean particle size; DLR, drug loading rate.

Table 6 Analysis of variance for MPS and DLR – reduced model

Source	MPS					Source	DLR
	Degrees of freedom	Sum of squares (SS)	Mean square (MS)	F	P-value		Degrees of freedom	Sum of squares (SS)	Mean square (MS)	F	P-value
X2	1	55793.98	55793.98	16.84	0.0027	X1	1	3798.87	3798.87	54.02	0.0000
X3	1	62196.62	62196.62	18.77	0.0019	X2	1	3785.94	3785.94	53.84	0.0000
X5	1	24181.03	24181.03	7.30	0.0243	X1 × X2	1	2698.28	2698.28	38.37	0.0000
X1 × X3	1	3.818E + 005	3.818E + 005	115.22	0.0000
X1 × X4	1	68417.56	68417.56	20.65	0.0014
X2 × X4	1	1.400E + 005	1.400E + 005	42.27	0.0001
X2 × X5	1	95504.18	95504.18	28.82	0.0005
X4 × X5	1	89567.02	89567.02	27.03	0.0006
Residual error	9	29819.65	3313.29			Residual error	14	984.54	70.32
Lack of fit	7	28736.20	4105.17	7.58	0.1215	Lack of fit	12	982.56	81.88	82.70	0.0120
Pure error	2	1083.45	541.73			Pure error	2	1.98	0.99
Total	18	9.699E + 005				Total	18	11349.74

Abbreviations: MPS, mean particle size; DLR, drug loading rate.

Figure 6 Main effects plot for (A) MPS and (B) DLR.

Abbreviations: MPS, mean particle size; DLR, drug loading rate.

Figure 7 Interaction plot for (A) MPS and (B) DLR.

Abbreviations: MPS, mean particle size; DLR, drug loading rate.

Figure 8 Normal probability plot of residual values for (A) MPS and (B) DLR.

Abbreviations: MPS, mean particle size; DLR, drug loading rate.

Figure 9 Comparison of SEM images of original Oli-A, FA-CS and FA-Oli-CSNPs. (A) Irregular lamelliform crystals of raw Oli-A particles. (B) FA-CS lyophilized powder. (C) FA-Oli-CSNPs dispersed with deionized water. (D) FA-Oli-CSNPs containing mannitol dispersed with deionized water.

Abbreviations: Oli-A, oligomycin-A; FA-CS, folate-conjugated chitosan; FA-Oli-CSNPs, folate-conjugated chitosan nanoparticles which encapsulate oligomycin-A.

Figure 10 The particle size distributions of FA-Oli-CSNPs which were determined by DLS. (A) FA-Oli-CSNPs dispersed with deionized water. (B) FA-Oli-CSNPs containing mannitol dispersed with deionized water.

Abbreviations: FA-Oli-CSNPs, folate-conjugated chitosan nanoparticles which encapsulate oligomycin-A; DLS, dynamic laser light scattering.

Figure 11 FA-Oli-CSNPs accumulative drug release profile.

Abbreviation: FA-Oli-CSNPs, folate-conjugated chitosan nanoparticles which encapsulate oligomycin-A.