Combinatorial therapeutic approach for treatment of oral squamous cell carcinoma

Figures & data

Table 1. Different composition of SLN formulation.

Formulation Code	Amount of drug(mg)	Amount of lipid (g)	Amount of Egg Lecithin (mg)	Amount of Pluronic F68 (g)
F1-P	10	0.5	10	1.0
F2-P	10	0.5	20	1.5
F3-P	10	0.5	30	2.0
F4-P	10	0.75	10	1.0
F5-P	10	0.75	20	1.5
F6-P	10	0.75	30	2.0
F7-P	–	1.0	10	1.0
F8-P	10	1.0	20	1.5
F9-P	10	1.0	30	2.0
F1-5FU	10	0.5	10	1.0
F2-5FU	10	0.5	20	1.5
F3-5FU	10	0.5	30	2.0
F4-5FU	10	0.75	10	1.0
F5-5FU	10	0.75	20	1.5
F6-5FU	10	0.75	30	2.0
F7-5FU	–	1.0	10	1.0
F8-5FU	10	1.0	20	1.5
F9-5FU	10	1.0	30	2.0
F1-AA	10	0.5	10	1.0
F2-AA	10	0.5	20	1.5
F3-AA	10	0.5	30	2.0
F4-AA	10	0.75	10	1.0
F5-AA	10	0.75	20	1.5
F6-AA	10	0.75	30	2.0
F7-AA	–	1.0	10	1.0
F8-AA	10	1.0	20	1.5
F9-AA	10	1.0	30	2.0

Table 2. In-vivo experimental design.

Serial Number	Group	Group Designation	Treatment
1	1	Normal Control	OSCC not induced
2	2	Negative Control	OSCC induced but not treated with SLN
3	3	Test	After induction of OSCC, treated with PTX SLN
4	4	Test	After induction of OSCC, treated with 5-FU SLN
5	5	Test	After induction of OSCC, treated with AA SLN
6	6	Test	After induction of OSCC, treated with both AA SLN and PTX SLN (Dose: 1:1)
7	7	Test	After induction of OSCC, treated with both AA SLN and 5-FU SLN (Dose: 1:1)
8	8	Test	After induction of OSCC, treated with both 5-FU SLN and PTX SLN (Dose: 1:1)

Table 3. Physicochemical evaluation of solid lipid nanoparticles.

Formulation Code	Particle Size (Z avg ± SD) (nm)	Polydispersity Index	Zeta Potential (mV ± SD)	Entrapment Efficiency %	% Drug Loading	% Yield
F1-P	399.1 ± 8.6	0.42	−35.9 ± 2.1	61.6	14.46	66.19
F2-P	268.4 ± 6.4	0.36	−32.4 ± 1.8	64.9	21.4	66.13
F3-P	78.82 ± 5.2	0.22	−33.8 ± 1.6	73.1	31.6	66.42
F4-P	469.9 ± 12.4	0.38	−34.0 ± 0.8	62.8	11.25	62.85
F5-P	397.9 ± 6.7	0.32	−32.1 ± 1.1	65.6	14.18	62.36
F6-P	242.4 ± 5.4	0.29	−33.6 ± 1.2	67.7	16.4	61.12
F7-P	482.1 ± 11.6	0.34	−25.2 ± 0.8	–	–	–
F8-P	532.6 ± 21.2	0.38	−30.8 ± 0.9	66.3	13.73	60.31
F9-P	587.8 ± 3.6	0.34	−31.4 ± 1.6	68.3	15.32	60.97
F1-5FU	412.1 ± 3.12	0.65	−30.1 ± 1.6	59.1	11.87	61.24
F2-5FU	389.8 ± 4.67	0.53	−31.6 ± 2.7	58.9	13.8	60.1
F3-5FU	328.7 ± 6.11	0.47	−31.8 ± 1.7	60.3	18.7	59.9
F4-5FU	386.9 ± 2.44	0.34	−31.3 ± 1.4	65.1	19.5	59.65
F5-5FU	312.5 ± 1.23	0.31	−32.2 ± 1.5	66.3	21.3	59.23
F6-5FU	117.5 ± 3.4	0.24	−32.9 ± 1.2	68.1	28.9	60.1
F7-5FU	418.3 ± 2.3	0.61	−30.1 ± 1.5	–	–	–
F8-5FU	487.1 ± 1.2	0.54	−30.3 ± 1.5	61.5	25.6	58.71
F9-5FU	511.6 ± 1.2	0.67	−28.8 ± 2.1	60.4	28.7	58.54
F1-AA	398.1 ± 2.3	0.54	−29.1 ± 1.2	57.1	16.3	61.26
F2-AA	387.3 ± 1.2	0.34	−30.1 ± 2.1	55.5	19.3	60.9
F3-AA	312.4 ± 4.6	0.45	−30.3 ± 1.2	58.7	22.4	60.16
F4-AA	402.1 ± 1.5	0.33	−31.2 ± 1.8	60.1	19.4	59.45
F5-AA	338.4 ± 2.7	0.44	−31.4 ± 1.4	61.4	21.9	59.15
F6-AA	143.1 ± 2.3	0.27	−31.7 ± 1.5	62.1	30.4	59.1
F7-AA	404.1 ± 2.6	0.56	−30.8 ± 1.7	–	–	–
F8-AA	423.1 ± 8.1	0.77	−30.4 ± 2.8	54.6	29.1	58.75
F9-AA	456.7 ± 3.2	0.67	−28.1 ± 2.4	54.4	28.5	58.2

Figure 1. Scanning Electron Micrograph of SLNs.

Figure 2. Transmission Electron Micrograph of SLNs.

Table 4. Release kinetics of SLN.

Formulation	Zero order r^2	First order r^2	Higuchi r^2	Korsemeyer-peppas r^2	n
F3-P	.967	.987	.989	.990	0.646
F6-5FU	.948	.975	.987	.984	0.543
F6-AA	.953	.986	.991	.971	0.509

Figure 3. Fourier transform infrared (FTIR) spectra of (a) PTX loaded SLN, (b) Fourier transform infrared (FTIR) spectra of 5-FU loaded SLN, (c) Fourier transform infrared (FTIR) spectra of AA loaded SLN.

Figure 4. Differential scanning calorimetry thermograms of (a) PTX loaded SLN, (b) Differential scanning calorimetry thermograms of 5-FU loaded SLN, (c) Differential scanning calorimetry thermograms of AA loaded SLN.

Table 5. Results of stability studies of SLN.

Temperature °C	Particle size (nm)		PV	Zeta Potential (mv)		PV	PDI		PV
	0 Month	3 Months		0 Month	3 Months		0 Month	3 Months
For PTX loaded SLN
4	78.82±5.2	79.44 ± 5.2	0.8	−33.8 ± 1.6	−32.2 ± 3.2	4.7	0.22	0.23	4.5
25	78.82±5.2	79.24 ± 5.4	1.4	−33.8 ± 1.6	−31.6 ± 4.2	6.5	0.22	0.24	9.0
For 5-FU loaded SLN
4	117.5 ± 3.4	119.43 ± 3.6	1.6	−32.9 ± 1.2	−31.1 ± 1.2	5.4	0.24	0.26	8.3
25	117.5 ± 3.4	119.78 ± 3.4	1.9	−32.9 ± 1.2	30.9 ± 1.6	6.1	0.24	0.27	12.5
For AA loaded SLN
4	143.1 ± 2.3	146.2 ± 2.4	2.1	−31.7 ± 1.5	30.2 ± 2.4	4.7	0.27	0.29	7.4
25	143.1 ± 2.3	146.8 ± 2.7	2.5	−31.7 ± 1.5	29.9 ± 2.7	5.6	0.27	0.30	11.1

PV: Percent Variation.

Figure 5. Histological changes in the mice tongue tissue after two weeks of treatment period.

Figure 6. Histological changes in the mice tongue tissue after four weeks of treatment period.

Figure 7. Pharmacokinetic profile of (a) PTX loaded SLN, (b) Pharmacokinetic profile of 5-FU loaded SLN, (c) Pharmacokinetic profile of AA loaded SLN.

Table 6. Results of pharmacokinetic studies of SLN.

Parameters	Units	PTX-C	PTX-T	5-FU-C	5-FU-T	AA-C	AA-T
Tmax	h	2.0 ± 0.0	6 ± 0.0***	2.0 ± 0.0	4 ± 0.0***	1.0 ± 0.0	2 ± 0.0***
Cmax	ng/ml	9823.4 ± 340.2	8523.61±370.8***	9431.45 ± 330.2	8902.42 ± 350.6*	9511.23 ± 348.9	8730.58 ± 339.5***
AUC0–24	ng.h/ml	20557.81 ± 147.8	71411.7 ± 170.7***	30914.16 ± 138.1	60156.16 ± 169.66***	29500.53 ± 138.4	50859.84 ± 143.2***
AUC0–∞	ng.h/ml	20758.06 ± 132.5	73215.57 ± 121.2***	31178.29 ± 121.4	61971.94 ± 132.1***	29761.92 ± 123.6	53326.63 ± 112.2***
MRT0–∞	h	2.5 ± 0.16	4.89 ± 0.32***	4.4 ± 0.21	4.73 ± 0.36	4.24 ± 0.32	5.6 ± 0.64**
Vd	L/kg	12.16 ± 1.1	6.15 ± 1.6 ***	14.23 ± 1.4	7.86 ± 1.3 ***	14.37 ± 1.7	2.02 ± 2.3***
CL	L h^−1 kg^−1	0.48 ± 0.03	0.12 ± 0.04 ***	0.32 ± 0.03	0.16 ± 0.04***	0.33 ± 0.02	0.19 ± 0.01***
Kel	h^−1	0.4 ± 0.01	0.204 ± 0.04***	0.22 ± 0.02	0.211 ± 0.01	0.23 ± 0.02	0.178 ± 0.04*
t1/2	h	1.7 ± 0.08	3.39 ± 0.13***	3.15 ± 0.58	3.26 ± 0.03	3.01 ± 0.53	3.8 ± 0.18*

In the above table, *represents p < .05.; **represents p < .01;***represents p < .0001.

Tmax: time taken to reach the maximum concentration of the drug in the plasma; Cmax: maximum concentration of drug in the plasma; AUC0–24: area under the concentration-time curve from zero up to 24h; AUC0–∞: area under the concentration-time curve from zero up to ∞ with extrapolation of the terminal phase; MRT0–∞: mean residence time of the unchanged drug in the systemic circulation from time zero up to ∞; Vd: volume of distribution; CL: total plasma clearance; Kel: elimination rate constant; t ½: elimination half-life or time taken for Cmax to drop in half.