A new approach for the management of Escherichia coli and Klebsiella pneumonia by using cefixime-based bionanocomposite films

Figures & data

Table 1. Formulation development design.

Formulation Codes	$\underset{100\mathrm{ }\text{parts}}{\underbrace{\text{Chitosan}:\mathrm{ }\text{Starch}}}$		Sepiolite (phr)	Cefixime (phr)	Glycerol (phr)
F1	100	0	3	20	50
F2	75	25	3	20	50
F3	50	50	3	20	50
F4	25	75	3	20	50
F5	0	100	3	20	50
F6	100	0	3	0	50
F7	75	25	3	0	50
F8	50	50	3	0	50
F9	25	75	3	0	50
F10	0	100	3	0	50
40kGy
F11	100	0	3	20	50
F12	75	25	3	20	50
F13	50	50	3	20	50
F14	25	75	3	20	50
F15	0	100	3	20	50
F16	100	0	3	0	50
F17	75	25	3	0	50
F18	50	50	3	0	50
F19	25	75	3	0	50
F20	0	100	3	0	50
80kGy
F21	100	0	3	20	50
F22	75	25	3	20	50
F23	50	50	3	20	50
F24	25	75	3	20	50
F25	0	100	3	20	50
F26	100	0	3	0	50
F27	75	25	3	0	50
F28	50	50	3	0	50
F29	25	75	3	0	50
F30	0	100	3	0	50

Note: phr stands for parts per hundred.

Figure 1. FTIR analysis of various formulations: (A) F3, (B) F13, (C) F23, (D) F8, (E) F18, (F) F28.

Table 2. FTIR absorption values of various formulations: (A) F3, (B) F13, (C) F23, (D) F8, (E) F18, (F) F28.

S. No	Functional groups				Observed values
	Chitosan	Starch	Sepiolite	Cefixime	F3	F13	F23	F8	F18	F28
1	-OH Stretching 3000–3750 cm^−1	-OH Stretching (3290 cm^−1)			3285.3 cm^−1	3285.2 cm^−1	3277.8 cm^−1′	3267.5 cm^−1	3278.3 cm^−1	3276.9 cm^−1
2	-C = O- Stretching (1480–1680 cm^−1)			-C = O Stretching amide (1667 cm^−1)	1638.6 cm^−1	1641.9 cm^−1	1636.6 cm^−1	1639.1 cm^−1	1640.3 cm^−1	1640.3 cm^−1
3	N-H Amide II bending 1550–1640 cm^−1			N-O Stretching (1515–1560 cm^−1)	1553.8 cm^−1	1536.0 cm^−1	1558.0 cm^−1	1557.6 cm^−1	1558.6 cm^−1	1558.4 cm^−1
4			-Si-O-Si- Stretching (980–1070 cm^−1)		923.21 cm^−1	921.12 cm^−1	925.34 cm^−1	925.15 cm^−1	926.60 cm^−1	924.52 cm^−1

Figure 2. TGA curves of non-drug and drug-loaded formulations of non-irradiated and γ-irradiated nanocomposite films.

Table 3. Thermal studies of non-drug and drug-loaded formulations of non-irradiated and γ-irradiated bionanocomposite films at different temperatures.

S. no.	Formulation codes	Mass loss (%) 150 °C	Mass loss (%) 250 °C	Mass loss (%) 350 °C	Residue (%) 550 °C
1	F8	18.31	37.823	74.03	14.81
2	F3	15.31	32.37	71.07	16.124
3	F18	20.53	46.95	74.0	17.32
4	F13	18.31	36.23	69.09	18.321
5	F28	17.01	34.123	67.147	17.562
6	F23	14.27	28.5952	54.134	18.321

Figure 3. SEM image of cefixime loaded formulation F23 irradiated with 80 kGy dose (a) at a magnification of 2500×, (b) at a magnification of 5000×, (c) at a magnification of 10,000×, (d) non-drug loaded (F8) at magnification of 10,000×.

Figure 4. EDX analysis of 80 kGy γ-irradiated formulation F23.

Table 4. Elemental composition of 80 kGy γ-irradiated formulation F23.

Element	Weight (%)
Carbon	39.48
Nitrogen	10.48
Oxygen	46.93
Magnesium	0.47
Silica	0.95
Sulphur	0.57
Calcium	0.75
Total	100

Table 5. Solubility studies of cefixime in different solvent systems at 37 ± 2 °C for 24 h.

Solvent system	Solubility (mg/ml) at 37 ± 2 °C
Acetone	10
Acetic acid	04
Acetonitrile	09
Chloroform	09
Ethanol	16
Ethyl acetate	03
Dichloromethane	06
Methanol	39
Propylene glycol	07
Water	02
Phosphate buffer saline pH 6.8	12
Phosphate buffer saline pH 7.0	14
Phosphate buffer saline pH 7.2	16
Phosphate buffer saline pH 7.4	20
Acetone: Phosphate buffer saline pH 7.4(50:50)	02
Ethanol: Phosphate buffer saline pH 7.4(50:50)	12
Methanol: Phosphate buffer saline pH 7.2(50:50)	08
Methanol: Phosphate buffer saline pH 7.4(50:50)	18
Methanol: Phosphate buffer saline pH 7.4(30:70)	10
Methanol: Phosphate buffer saline pH 7.4(40:60)	10

Figure 5. Standard calibration curve of cefixime made with series of dilutions ranging from 1 µg/ml to 10 µg/ml.

Table 6. Drug content uniformity/drug loading efficiency of formulations.

Formulation code	Contents in centre (%± S.D)	Contents in proximity (% ± S.D)
F1	99.450 ± 0.72	99.250 ± 0.98
F2	99.705 ± 1.80	99.456 ± 1.76
F3	98.173 ± 1.08	98.156 ± 1.03
F4	96.896 ± 3.61	96.891 ± 3.12
F5	93.065 ± 1.08	93.078 ± 1.10
F11	94.087 ± 0.36	94.081 ± 0.40
F12	97.151 ± 1.08	97.156 ± 1.04
F13	97.918 ± 1.44	97.912 ± 1.37
F14	98.428 ± 2.16	98.421 ± 2.12
F15	97.662 ± 1.08	97.668 ± 1.03
F21	97.407 ± 1.44	97.401 ± 1.50
F22	98.939 ± 1.44	98.931 ± 1.48
F23	96.385 ± 2.88	96.381 ± 2.83
F24	99.195 ± 3.25	99.198 ± 3.27
F25	98.939 ± 2.16	98.940 ± 2.18

Figure 6. The swelling ratio of chitosan-starch nanocomposite films in (A) HCl buffer pH 1.2, (B) Acetate buffer pH 4.5, and (C) Phosphate buffer pH 6.8.

Figure 7. Percentage water content of chitosan-starch nanocomposite films in (A) HCl buffer pH 1.2, (B) Acetate buffer pH 4.5, and (C) Phosphate buffer pH 6.8.

Table 7. Percentage matrix erosion of non-irradiated and γ-irradiated bionanocomposite films at pH 1.2; 4.5 and 6.8.

	Erosion (%± S.D.)
Formulation codes	pH 1.2	pH 4.5	pH 6.8
F1	36.9 ± 2.12	31.3 ± 3.35	29.5 ± 0.99
F2	35.2 ± 5.51	30.6 ± 1.27	27.8 ± 3.68
F3	40.5 ± 0.56	33.9 ± 1.69	31.4 ± 6.10
F4	34.1 ± 4.81	26.4 ± 0.99	24.0 ± 0.85
F5	30.4 ± 1.56	25.6 ± 0.57	23.3 ± 0.85
F11	35.5 ± 2.83	30.7 ± 0.28	26.9 ± 1.71
F12	34.2 ± 1.27	29.3 ± 0.27	25.1 ± 3.94
F13	38.4 ± 2.55	31.5 ± 2.69	29.6 ± 0.42
F14	32.6 ± 2.97	25.1 ± 4.10	24.4 ± 2.69
F15	30.7 ± 5.52	24.7 ± 0.42	22.7 ± 2.97
F21	32.1 ± 2.10	27.3 ± 1.98	24.2 ± 1.56
F22	29.6 ± 2.57	25.4 ± 1.56	23.4 ± 1.98
F23	33.7 ± 1.56	28.5 ± 2.40	25.6 ± 2.26
F24	27.9 ± 2.14	22.9 ± 0.28	21.7 ± 2.12
F25	25.4 ± 1.25	21.3 ± 1.95	20.9 ± 2.97

Figure 8. (A) In vitro permeation profile of non-irradiated cefixime nanobiocomposite films; (B) 40kGy γ-irradiated cefixime nanobiocomposite films; (C) 80 kGy γ-irradiated cefixime nanocomposite films.

Table 8. Ex vivo skin permeation kinetic data of cefixime from both γ-irradiated and non-irradiated films.

Formulation codes	Flux (µg/cm^2/min)	D (Cm^2/min)	Kp (cm/min)
F1	1.7494 ± 0.13	0.0244 ± 7.3 × 10^−3	0.00049 ± 3.5 × 10^−5
F2	1.9705 ± 0.26	0.0246 ± 2.4 × 10^−3	0.00057 ± 7.5 × 10^−5
F3	2.6874 ± 0.26	0.0214 ± 3.8 × 10^−3	0.00073 ± 6.8 × 10^−5
F4	3.3345 ± 0.14	0.0227 ± 1.5 × 10^−3	0.00086 ± 3.5 × 10^−5
F5	5.0257 ± 0.29	0.0219 ± 1.8 × 10^−3	0.00122 ± 7.1 × 10^−5
F11	1.6088 ± 0.14	0.0299 ± 3.0 × 10^−3	0.00045 ± 3.7 × 10^−5
F12	1.9086 ± 0.08	0.0174 ± 2.9 × 10^−3	0.00056 ± 2.4 × 10^−5
F13	2.1714 ± 0.18	0.0076 ± 2.3 × 10^−3	0.00059 ± 4.9 × 10^−5
F14	2.3930 ± 0.26	0.0047 ± 3.0 × 10^−3	0.00061 ± 6.6 × 10^−5
F15	2.7467 ± 0.15	0.0117 ± 2.7 × 10^−3	0.00067 ± 3.6 × 10^−5
F21	1.0711 ± 0.09	0.0101 ± 1.8 × 10^−3	0.00028 ± 2.4 × 10^−5
F22	1.3852 ± 0.08	0.0088 ± 3.4 × 10^−3	0.00040 ± 2.4 × 10^−5
F23	1.8245 ± 0.51	0.0222 ± 2.6 × 10^−3	0.00049 ± 1.3 × 10^−5
F24	2.0185 ± 0.03	0.0208 ± 2.7 × 10^−3	0.00051 ± 7.9 × 10^−6
F25	2.2162 ± 0.06	0.0182 ± 1.2 × 10^−3	0.00054 ± 1.5 × 10^−5

Figure 9. (A) Release behavior of non-irradiated nanocomposite films, (B) γ-irradiated films at a dose of 40 kGy, (C) γ-irradiated films at a dose of 80 kGy.

Table 9. Kinetic profile of γ-irradiated and non-irradiated bionanocomposite films.

	Zero order model			First order model		Higuchi model		Korsmeyer-Peppas model
Formulation codes	k0	R^2	t25%	K0	R^2	K0	R^2	KKP	R^2	N
F1	2.575	0.9448	9.711	0.035	0.9888	9.702	0.9094	4.966	0.9821	0.763
F2	2.837	0.9035	8.812	0.040	0.9803	10.870	0.9601	7.193	0.9951	0.664
F3	3.051	0.8771	8.194	0.045	0.9709	11.755	0.9708	8.459	0.9950	0.631
F4	3.788	0.9251	6.599	0.061	0.9975	14.446	0.9523	8.885	0.9971	0.692
F5	4.437	0.8509	5.634	0.083	0.9963	17.145	0.9593	12.632	0.9810	0.621
F11	1.511	0.8624	16.541	0.018	0.9166	5.817	0.9431	4.045	0.9720	0.644
F12	1.825	0.9309	13.701	0.022	0.9726	6.928	0.9331	3.979	0.9884	0.719
F13	2.214	0.9571	11.294	0.028	0.9909	8.331	0.9144	4.153	0.9906	0.773
F14	2.859	0.9563	8.744	0.040	0.9923	10.715	0.8946	4.997	0.9817	0.799
F15	3.359	0.9243	7.442	0.051	0.9913	12.729	0.9157	7.080	0.9762	0.731
F21	1.385	0.9125	18.055	0.016	0.9480	5.285	0.9437	3.288	0.9871	0.688
F22	1.580	0.8701	15.281	0.019	0.9245	6.090	0.963	4.362	0.9881	0.633
F23	1.916	0.7984	13.046	0.024	0.8848	7.465	0.9658	6.131	0.9760	0.579
F24	2.274	0.8101	10.994	0.030	0.9114	8.847	0.9681	7.138	0.9800	0.586
F25	2.730	0.8568	9.156	0.038	0.9569	10.538	0.958	7.658	0.98170	0.627

Table 10. Comparison of similarity factor (f₂) of different formulations.

Comparison formulation	Similarity factor (f2) values	Comparison formulation	Similarity factor (f2) values
F1 vs. F2	69.60	F1 vs. F22	48.29
F1 vs. F3	59.68	F1 vs. F23	56.29
F1 vs. F4	43.55	F1 vs. F24	68.03
F1 vs. F5	33.76	F1 vs. F25	72.76
F1 vs. F11	46.98	F11 vs. F21	83.03
F1 vs.F12	54.33	F12 vs. F22	76.48
F1 vs. F13	68.52	F13 vs. F23	69.12
F1 vs. F14	72.40	F14 vs. F24	57.02
F1 vs. F15	52.81	F15 vs. F25	57.33
F1 vs. F21	44.43

Table 11. Antibacterial activities of different bionanocomposite films (γ-irradiated and non-irradiated) against different bacteria.

	Sample plate		40 kGy plate		80 kGy plate
	Formulation code	Zone of inhibition (mm)	Formulation code	Zone of inhibition (mm)	Formulation code	Zone of inhibition (mm)
Activity against Klebsiella pneumonia	F1	10	F11	10	F21	Nil
	F2	15	F12	18	F22	Nil
	F3	17	F13	12	F23	Nil
	F4	20	F14	Nil	F24	Nil
	F5	25	F15	Nil	F25	9
	N	Nil	N	Nil	N	Nil
	Standard	30	Standard	28	Standard	27
	Sample plate		40 kGy plate		80 kGy plate
	Formulation code	Zone of inhibition (mm)	Formulation code	Zone of inhibition (mm)	Formulation code	Zone of inhibition (mm)
Activity against Acetobacter aceti	F1	18	F11	Nil	F21	Nil
	F2	29	F12	14	F22	10
	F3	21	F13	Nil	F23	Nil
	F4	15	F14	Nil	F24	Nil
	F5	13	F15	20	F25	Nil
	N	Nil	N	Nil	N	Nil
	Standard	25	Standard	20	Standard	28
	Sample plate		40 kGy plate		80 kGy plate
	Formulation code	Zone of inhibition (mm)	Formulation code	Zone of inhibition (mm)	Formulation code	Zone of inhibition (mm)
Activity against Escherichia coli	F1	Nil	F11	10	F21	10
	F2	9	F12	12	F22	9
	F3	12	F13	Nil	F23	Nil
	F4	13	F14	Nil	F24	Nil
	F5	20	F15	Nil	F25	Nil
	N	Nil	N	Nil	N	Nil
	Standard	30	Standard	30	Standard	30

Note: N = Negative control i.e. DMSO (dimethyl sulfoxide).

Figure 10. Antimicrobial activities of γ-irradiated and non-irradiated bionanocomposite films against (A) Escherichia coli, (B) Klebsiella pneumonia, and (C) Acetobacter aceti.

Data availability

All the data have been added to this article.