RETRACTED ARTICLE: PNIPAM nanoparticles for targeted and enhanced nose-to-brain delivery of curcuminoids: UPLC/ESI-Q-ToF-MS/MS-based pharmacokinetics and pharmacodynamic evaluation in cerebral ischemia model

Figures & data

Table 1. Optimization of placebo NPs (PNIPAM-NPs) on the basis of NIPAM, VP, AA and MBA ratio.

Formulation code	NIPAM (%)	VP (%)	AA (%)	Concentration of MBA (mg/mL)	Mean particle size (nm ± SD)	Mean PDI ± SD	PY (%) ± SD
A1	50	25	25	20	815.9 ± 212.1	0.906 ± 0.048	21.78 ± 1.75
A2	50	15	35	20	989.8 ± 115.3	0.989 ± 0.032	17.34 ± 2.35
A3	50	35	15	20	727.4 ± 106.6	0.856 ± 0.028	23.73 ± 1021
B1	60	20	20	20	415.1 ± 66.8	0.325 ± 0.045	27.34 ± 2.45
B2	60	15	25	20	519.2 ± 45.7	0.336 ± 0.033	35.54 ± 1.76
B3	60	25	15	20	489.2 ± 59.9	0.346 ± 0.029	38.46 ± 2.12
C1	70	15	15	20	268.3 ± 27.7	0.291 ± 0.022	42.56 ± 2.33
C2	70	10	20	20	288.9 ± 21.8	0.286 ± 0.018	41.61 ± 3.46
C3	70	20	10	20	232.6 ± 22.3	0.281 ± 0.015	48.63 ± 5.16
D1	80	10	10	20	88.6 ± 45	0.272 ± 0.028	82.90 ± 3.78
D2	80	5	15	20	108.3 ± 3.8	0.189 ± 0.029	80.24 ± 1.17
D3	80	15	5	20	82.4 ± 5.3	0.106 ± 0.039	72.24 ± 2.13

Figure 1. Comparative SEM (left) and TEM (right) images of (A and D) PNIPAM-CUR, (B and E) PNIPAM-DMC and (C and F) PNIPAM-BDMC.

Table 2. Optimization of drug polymer ratio of CUR-PNIPAM, DMC-PNIPAM and BDMC-PNIPAM on the basis of particle size, PDI, zeta potential, PY, LC and EE (%).

Formulation code	Drug:polymer ratio	Mean particle size (nm ± SD)	Mean PDI ± SD	PY ± SD	Mean zeta potential (mV ± SD)	LC (%)	EE (%)
CUR-PNIPAM-1	1:10	92.46 ± 2.8	0.191 ± 0.052	81.83 ± 4.69	−16.2 ± 1.42	39.31 ± 3.7	84.63 ± 4.2
CUR-PNIPAM-2	2:10	198.74 ± 6.6	0.423 ± 0.042	83.29 ± 5.24	−15.02 ± 2.90	51.73 ± 2.4	81.24 ± 4.9
CUR-PNIPAM-3	3:10	328.24 ± 8.9	0.462 ± 0.064	87.23 ± 4.78	−12.1 ± 3.16	59.29 ± 2.8	74.13 ± 3.9
DMC-PNIPAM-1	1:10	91.23 ± 4.2	0.183 ± 0.063	82.06 ± 4.29	−15.6 ± 1.33	38.91 ± 3.6	84.71 ± 3.99
DMC-PNIPAM-2	2:10	196.78 ± 6.2	0.429 ± 0.049	84.91 ± 6.11	−14.9 ± 2.78	52.16 ± 2.9	80.33 ± 4.2
DMC-PNIPAM-3	3:10	337.63 ± 7.2	0.469 ± 0.061	86.73 ± 4.62	−13.6 ± 3.76	62.39 ± 3.4	71.23 ± 2.6
BDMC-PNIPAM-1	1:10	94.28 ± 1.91	0.142 ± 0.046	82.29 ± 4.71	−16.6 ± 1.21	40.61 ± 3.6	85.73 ± 4.31
BDMC-PNIPAM-2	2:10	205.53 ± 3.16	0.424 ± 0.046	86.74 ± 2.12	−14.3 ± 2.69	53.23 ± 2.9	82.16 ± 4.62
BDMC-PNIPAM-3	3:10	346.23 ± 6.9	0.472 ± 0.068	88.24 ± 4.18	−12.8 ± 3.27	65.26 ± 4.2	72.93 ± 3.9

SD, standard deviation. All the above nine formulation PNIPAM -NPs, PNIPAM/MBA ratio fixed at 99.4/0.6 in which NIPAM/VP/AA::85/10/05.

Figure 2. NMR spectra of the developed co-poly-(NIPAM–VP–AA) revealing the formation of PNIPAM.

Figure 3. DSC thermograms of pure CUR, DMC and BDMC, physical mixtures of monomers and curcuminoids and freeze-dried NPs.

Figure 4. The cumulative percentage release of CUR, DMC, BDMC from CUR-PNIPAM, DMC-PNIPAM and BDMC-PNIPAM (drug:polymer ratio 10:1) as compared to pure CUR, DMC and BDMC.

Figure 5. Ex vivo permeation profiles of developed NPs as compared to pure curcuminoids through goat nasal mucosa.

Figure 6. MS and MSMS scans of CUR, DMC, BDMC and IS (nimesulide).

Figure 7. Typical chromatograms of brain homogenate extracted (A) CUR, (B) DMC, (C) BDMC, (D) Blank and (E) Nimesulide (IS).

Table 3. Validation: precision and accuracy data for CUR in different biomatrixes.

			Intra-batch precision			Inter-batch precision
Biomatrix	Quality controls samples	Theoretical concentration (ng mL^−1 or ng g^−1)	Observed concentration (ng mL^−1 or ng^−1) ± SD	Accuracy (%)	Precision (%C.V.)	Observed concentration (ng mL^−1 or ng g^−1) ± SD	Accuracy (%)	Precision (%C.V.)	Recovery (%)
Brain  homogenate	LLOQQC	1.00	0.97 ± 0.011	96.29	1.17	0.97 ± 0.012	96.33	1.21	86.79
	LQC	2.90	2.83 ± 0.062	97.41	2.20	2.84 ± 0.050	98.02	1.74	87.96
	MQC	400.00	393.50 ± 3.820	98.37	0.97	393.05 ± 3.925	98.26	1.00	89.10
	HQC	800.00	792.60 ± 3.741	99.08	0.47	792.53 ± 4.132	99.07	0.52	89.16
Lungs  homogenate	LLOQQC	1.00	0.95 ± 0.014	95.00	1.49	0.94 ± 0.02	93.5	2.27	87.67
	LQC	2.90	2.79 ± 0.035	96.03	1.27	2.78 ± 0.014	95.86	0.51	88.19
	MQC	400.00	390.5 ± 3.56	97.63	0.91	390.25 ± 0.354	97.56	0.09	89.27
	HQC	800.00	793.5 ± 2.12	99.19	0.27	791.75 ± 2.475	98.97	0.31	89.79
Plasma	LLOQQC	1.00	0.925 ± 0.035	92.5	3.82	0.93 ± 0.007	93.00	0.76	81.42
	LQC	2.90	2.73 ± 0.057	94.14	2.07	2.75 ± 0.028	94.83	1.03	95.30
	MQC	400.00	379.50 ± 2.121	94.88	0.56	382.75 ± 4.596	95.69	1.20	94.12
	HQC	800.00	783.5 ± 3.54	97.94	0.45	791.75 ± 2.475	98.97	0.31	92.32

Table 4. Validation: precision and accuracy data for DMC in different biomatrixes.

			Intra-batch precision			Inter-batch precision
Biomatrix	Quality controls samples	Theoretical concentration (ng mL^−1 or ng g^−1)	Observed concentration (ng mL^−1 or ng g^−1) ± SD	Accuracy (%)	Precision (%C.V.)	Observed concentration (ng mL^−1 or ng g^−1) ± SD	Accuracy (%)	Precision (%C.V.)	Recovery (%)
Brain  homogenate	LOQQC	1.00	0.96 ± 0.013	95.38	1.36	0.97 ± 0.013	95.87	1.30	87.67
	LQC	2.90	2.82 ± 0.047	97.18	1.65	2.84 ± 0.042	97.90	1.47	87.54
	MQC	400.00	393.46 ± 3.807	98.36	0.97	392.53 ± 3.663	98.13	0.93	89.33
	HQC	800.00	792.60 ± 3.690	99.03	0.47	792.24 ± 4.063	99.03	0.51	88.86
Lungs  homogenate	LOQQC	1.00	0.95 ± 0.021	94.5	2.24	0.94 ± 0.014	94.00	1.50	83.23
	LQC	2.90	2.80 ± 0.035	96.38	1.26	2.79 ± 0.021	96.03	0.76	86.28
	MQC	400.00	390.79 ± 2.12	97.63	0.54	390.25 ± 0.354	97.56	0.09	89.44
	HQC	800.00	792.25 ± 3.54	99.06	0.45	791.25 ± 1.77	98.91	0.22	89.85
Plasma	LOQQC	1.00	0.93 ± 0.028	93.00	3.04	0.93 ± 0.007	93.00	0.76	88.76
	LQC	2.90	2.74 ± 0.078	94.31	2.84	2.75 ± 0.028	94.83	1.03	88.92
	MQC	400.00	378.50 ± 2.121	94.63	0.56	382.75 ± 4.59	95.69	1.20	89.14
	HQC	800.00	781.00 ± 2.83	97.63	0.36	791.75 ± 2.475	98.97	0.31	89.93

Table 5. Validation: precision and accuracy data for BDMC in different biomatrixes.

			Intra-batch precision			Inter-batch precision
Biomatrix	Quality controls samples	Theoretical concentration (ng mL^−1 or ng g^−1)	Observed concentration (ng mL^−1 or ng g^−1) ± SD	Accuracy (%)	Precision (%C.V.)	Observed concentration (ng mL^−1 or ng g^−1) ± SD	Accuracy (%)	Precision (%C.V.)	Recovery (%)
Brain homogenate	LOQQC	1.00	0.95 ± 0.026	94.22	2.72	0.96 ± 0.019	94.72	1.97	87.34
	LQC	2.90	2.84 ± 0.036	97.82	1.27	2.84 ± 0.033	98.05	1.17	87.97
	MQC	400.00	392.58 ± 3.419	98.15	0.87	392.33 ± 3.560	98.08	0.91	89.09
	HQC	800.00	792.25 ± 3.455	99.03	0.44	792.51 ± 3.641	99.06	0.46	89.59
Lungs homogenate	LOQQC	1.00	0.94 ± 0.028	94.00	3.01	0.93 ± 0.014	93.00	1.52	82.97
	LQC	2.90	2.80 ± 0.014	96.55	0.51	2.79 ± 0.021	96.03	0.76	88.32
	MQC	400.00	389.5 ± 2.121	97.38	0.54	389.57 ± 0.354	97.44	0.09	87.95
	HQC	800.00	792.50 ± 3.54	99.06	0.45	791.25 ± 1.768	98.91	0.22	88.67
Plasma	LOQQC	1.00	0.915 ± 0.035	91.50	3.86	0.94 ± 0.014	92.50	1.53	80.26
	LQC	2.90	2.77 ± 0.035	95.34	1.28	2.78 ± 0.064	95.69	2.29	86.42
	MQC	400.00	384.5 ± 3.456	96.125	0.92	385.25 ± 1.061	96.31	0.28	81.82
	HQC	800.00	782.5 ± 2.12	97.81	0.27	785.25 ± 3.889	98.16	0.50	87.06

Table 6. Validation: stability data for CUR in different biomatrixes.

	LQC (2.90 ng/mL or ng g^−1)			MQC (400 ng/mL or ng g^−1)			HQC (800 ng/mL or ng g^-1)
Exposure condition	Brain homogenate	Lungs homogenate	Plasma	Brain homogenate	Lungs homogenate	Plasma	Brain homogenate	Lungs homogenate	Plasma
Long-term stability; recovery (ng) after storage (−80 °C)
Previous day	2.82 ± 0.03	2.79 ± 0.035	2.73 ± 0.057	393.50 ± 3.820	390.5 ± 3.56	379.50 ± 2.121	797.93 ± 1.40	793.53 ± 2.12	783.55 ± 3.541
Thirtieth day	2.80 ± 0.03 (99.29%)	2.77 ± 0.043 (99.28%)	2.72 ± 1.412 (99.63%)	389.23 ± 3.191 (98.91%)	385.42 ± 2.89 (98.70%)	376.14 ± 2.045 (99.11%)	788.37 ± 2.36 (98.80%)	783.91 ± 2.191 (98.79%)	780.94 ± 2.141 (99.67%)
Freeze-thaw stress; recovery (ng) after freeze-thaw cycles (−80 to 25 °C)
Pre-cycle	2.82 ± 0.03	2.79 ± 0.035	2.73 ± 0.057	393.50 ± 3.820	390.5 ± 3.56	379.50 ± 2.121	797.93 ± 1.40	793.53 ± 2.122	783.55 ± 3.541
First cycle	2.81 ± 0.03 (99.65%)	2.78 ± 0.025 (99.64%)	2.73 ± 1.151 (100%)	392.94 ± 2.791 (99.86%)	389.90 ± 2.46 (99.85%)	378.12 ± 1.210 (99.64%)	795.67 ± 1.59 (99.72%)	790.12 ± 1.493 (99.57%)	781.92 ± 1.491 (99.79%)
Second cycle	2.79 ± 0.02 (98.94%)	2.78 ± 0.012 (99.64%)	2.72 ± 1.211 (99.63%)	391.16 ± 1.141 (99.41)	388.79 ± 1.46 (99.56%)	377.49 ± 2.018 (99.47%)	794.80 ± 1.15 (99.61%)	788.79 ± 1.791 (99.40%)	778.91 ± 2.091 (99.41%)
Third cycle	2.76 ± 0.01 (97.87%)	2.77 ± 0.051 (99.28%)	2.71 ± 2.001 (99.27%)	390.02 ± 1.213 (99.12%)	388.19 ± 1.49 (99.41%)	376.91 ± 1.791 (99.32%)	791.32 ± 2.29 (99.17%)	783.146 ± 2.492 (98.69%)	775.11 ± 1.791 (98.92%)
Bench-top stability; recovery (ng) at room temperature (25 °C)
0 h	2.82 ± 0.03	2.79 ± 0.035	2.73 ± 0.057	393.50 ± 3.820	390.5 ± 3.56	379.50 ± 2.121	797.93 ± 1.40	793.53 ± 2.12	783.55 ± 3.541
24 h	2.81 ± 0.02 (99.65%)	2.77 ± 0.049 (99.28%)	2.72 ± 1.718 (99.63%)	391.15 ± 2.746 (99.40%)	389.04 ± 2.94 (99.63%)	378.09 ± 1.952 (99.63%)	795.29 ± 0.38 (99.67%)	786.491 ± 2.791 (99.11%)	777.12 ± 2.491 (98.92%)
Post-processing stability; recovery (ng) after storage in auto-sampler (4 °C)
0 h	2.82 ± 0.03	2.79 ± 0.035	2.73 ± 0.057	393.50 ± 3.820	390.5 ± 3.56	379.50 ± 2.121	797.93 ± 1.40	793.53 ± 2.12	783.55 ± 3.541
4 h	2.81 ± 0.02 (99.65%)	2.78 ± 0.028 (99.64%)	2.72 ± 0.091 (99.63%)	391.59 ± 2.491 (99.51%)	388.94 ± 1.29 (99.60%)	378.91 ± 1.107 (99.84%)	796.11 ± 1.06 (99.77%)	787.194 ± 1.927 (99.20%)	780.16 ± 2.121 (99.57%)

Values (mean ± SD) are derived from six replicates. Figures in parenthesis represent analyte concentration (%) relative to time zero. Theoretical contents: LQC: 2.9, MQC: 400 and HQC: 800 ng mL⁻¹.

Table 7. Validation: stability data for DMC in different biomatrixes.

	LQC (2.90 ng/mL or ng g^−1)			MQC (400 ng/mL or ng g^−1)			HQC (800 ng/mL or ng g^−1)
Exposure condition	Brain homogenate	Lungs homogenate	Plasma	Brain homogenate	Lungs homogenate	Plasma	Brain homogenate	Lungs homogenate	Plasma
Long-term stability; recovery (ng) after storage (−80 °C)
Previous day	2.85 ± 0.02	2.80 ± 0.035	2.74 ± 0.078	393.46 ± 3.807	390.79 ± 2.12	378.50 ± 2.121	795.68 ± 3.12	792.25 ± 3.54	781.00 ± 2.83
Thirtieth day	2.79 ± 0.03 (97.89%)	2.75 ± 0.041 (98.21%)	2.70 ± 1.121 (98.54%)	387.73 ± 2.791 (98.54%)	388.11 ± 1.711 (99.31)	375.13 ± 2.046 (99.11%)	786.46 ± 1.57 (98.84%)	785.91 ± 3.419 (99.20%)	778.06 ± 2.491 (99.62%)
Freeze-thaw stress; recovery (ng) after freeze-thaw cycles (−80 to 25 °C)
Pre-cycle	2.85 ± 0.02	2.80 ± 0.035	2.74 ± 0.078	393.46 ± 3.807	390.79 ± 2.12	378.50 ± 2.121	795.68 ± 3.12	792.25 ± 3.54	781.00 ± 2.83
First cycle	2.82 ± 0.02 (98.95%)	2.79 ± 0.0431 (99.64%)	2.73 ± 0.071 (99.64%)	391.13 ± 2.194 (99.41%)	389.17 ± 1.913 (99.59%)	377.03 ± 1.034 (99.61%)	791.55 ± 3.08 (99.48%)	790.37 ± 1.416 (99.76%)	779.93 ± 1.613 (99.86%)
Second cycle	2.79 ± 0.02 (97.89%)	2.77 ± 1.011 (98.93%)	2.71 ± 0.070 (98.91%)	389.19 ± 1.791 (98.91%)	388.13 ± 1.712 (99.32%)	376.49 ± 1.034 (99.47%)	788.74 ± 2.32 (99.13%)	788.13 ± 2.016 (99.48)	778.03 ± 1.037 (99.62%)
Third cycle	2.76 ± 0.01 (96.84%)	2.76 ± 1.104 (98.57%)	2.70 ± 0.072 (98.54%)	387.19 ± 1.919 (98.41%)	387.18 ± 1.818 (99.08%)	374.16 ± 1.161 (98.85%)	786.97 ± 2.85 (98.91%)	786.14 ± 1.037 (99.23%)	776.02 ± 2.709 (99.36%)
Bench-top stability; recovery (ng) at room temperature (25 °C)
0 h	2.85 ± 0.02	2.80 ± 0.035	2.74 ± 0.078	393.46 ± 3.807	390.79 ± 2.12	378.50 ± 2.121	795.68 ± 3.12	792.25 ± 3.54	781.00 ± 2.83
24 h	2.77 ± 0.02 (97.19%)	2.74 ± 0.094 (97.86%)	2.70 ± 0.079 (98.54%)	388.57 ± 2.716 (98.76%)	386.99 ± 1.146 (99.03%)	375.66 ± 1.312 (99.25%)	786.81 ± 2.29 (98.89%)	785.94 ± 2.161 (99.20%)	778.12 ± 1.373 (99.63%)
Post-processing stability; recovery (ng) after storage in auto-sampler (4 °C)
0 h	2.85 ± 0.02	2.80 ± 0.035	2.74 ± 0.078	393.46 ± 3.807	390.79 ± 2.12	378.50 ± 2.121	795.68 ± 3.12	792.25 ± 3.54	781.00 ± 2.83
24 h	2.81 ± 0.04 (98.60%)	2.77 ± 0.471 (98.93%)	2.72 ± 0.074 (99.27%)	390.16 ± 1.161 (99.16%)	388.93 ± 1.819 (99.52%)	376.31 ± 1.493 (99.42%)	794.73 ± 4.23 (99.88%)	789.96 ± 1.037 (99.71%)	779.19 ± 1.033 (99.77%)

Values (mean ± SD) are derived from six replicates. Figures in parenthesis represent analyte concentration (%) relative to time zero. Theoretical contents: LQC: 2.9; MQC: 400 and HQC: 800 ng mL⁻¹.

Table 8. Validation: stability data for BDMC in different biomatrixes.

	LQC (2.90 ng/mL or ng g^−1)			MQC (400 ng/mL or ng g^−1)			HQC (800 ng/mL or ng g^−1)
Exposure condition	Brain homogenate	Lungs homogenate	Plasma	Brain homogenate	Lungs homogenate	Plasma	Brain homogenate	Lungs homogenate	Plasma
Long-term stability; recovery (ng) after storage (−80 °C)
Previous day	2.86 ± 0.01	2.80 ± 0.014	2.77 ± 0.035	392.58 ± 3.419	389.5 ± 2.121	384.5 ± 3.456	795.69 ± 2.90	792.50 ± 3.54	782.5 ± 2.12
Thirtieth day	2.78 ± 0.03 (97.20%)	2.74 ± 0.046 (97.86%)	2.71 ± 0.049 (97.83%)	379.55 ± 2.488 (96.68%)	377.12 ± 2.790 (96.82%)	372.14 ± 2.791 (96.79%)	785.73 ± 1.48 (98.75%)	780.15 ± 2.761 (98.44%)	771.23 ± 3.106 (98.56%)
Freeze-thaw stress; recovery (ng) after freeze-thaw cycles (−80 to 25 °C)
Pre-cycle	2.86 ± 0.01	2.80 ± 0.014	2.77 ± 0.035	392.58 ± 3.419	389.5 ± 2.121	384.5 ± 3.456	795.69 ± 2.90	792.50 ± 3.54	782.5 ± 2.12
First cycle	2.84 ± 0.01 (99.30%)	2.78 ± 0.046 (99.29%)	2.75 ± 0.041 (99.28%)	388.14 ± 1.021 (98.87%)	387.19 ± 1.731 (99.41%)	381.76 ± 1.216 (99.29%)	792.99 ± 3.05 (99.66%)	788.19 ± 1.379 (99.46%)	777.35 ± 1.067 (99.34%)
Second cycle	2.82 ± 0.02 (98.60%)	2.75 ± 0.079 (98.21%)	2.72 ± 0.071 (98.19%)	384.19 ± 1.079 (97.86%)	385.08 ± 1.911 (98.87%)	378.91 ± 1.659 (98.55%)	789.85 ± 3.33 (99.27%)	782.13 ± 1.761 (98.69%)	774.03 ± 2.309 (98.92%)
Third cycle	2.79 ± 0.03 (97.55%)	2.73 ± 1.033 (97.50%)	2.70 ± 0.016 (97.47%)	379.13 ± 1.491 (96.57%)	378.81 ± 1.611 (97.26%)	373.192 ± 2.10 (97.06%)	786.24 ± 2.48 (98.81%)	779.46 ± 2.731 (98.35%)	771.34 ± 2.673 (98.57%)
Bench-top stability; recovery (ng) at room temperature (25 °C)
0 h	2.86 ± 0.01	2.80 ± 0.014	2.77 ± 0.035	392.58 ± 3.419	389.5 ± 2.121	384.5 ± 3.456	795.69 ± 2.90	792.50 ± 3.54	782.5 ± 2.12
24 h	2.79 ± 0.04 (97.55%)	2.72 ± 0.079 (97.14%)	2.71 ± 0.036 (97.83%)	378.99 ± 1.094 (96.54%)	377.81 ± 1.412 (97.00%)	372.91 ± 1.093 (96.99%)	787.12 ± 2.24 (98.92%)	779.91 ± 1.316 (98.41%)	772.93 ± 2.641 (98.78%)
Post-processing stability; recovery (ng) after storage in auto-sampler (4 °C)
0 h	2.86 ± 0.01	2.80 ± 0.014	2.77 ± 0.035	392.58 ± 3.419	389.5 ± 2.121	384.5 ± 3.456	795.69 ± 2.90	792.50 ± 3.54	782.5 ± 2.12
24 h	2.85 ± 0.01 (99.65%)	2.77 ± 0.46 (98.93%)	2.72 ± 0.070 (98.19%)	382.16 ± 2.716 (97.35%)	383.19 ± 1.614 (98.38%)	378.03 ± 2.011 (98.32%)	794.32 ± 1.90 (99.83%)	781.06 ± 1.694 (98.56%)	772.16 ± 2.046 (98.68%)

Values (mean ± SD) are derived from six replicates. Figures in parenthesis represent analyte concentration (%) relative to time zero. Theoretical contents: LQC: 2.9, MQC: 400 and HQC: 800 ng mL⁻¹.

Figure 8. Pharmacokinetic profiles of CUR, DMC and BDMC concentration in brain at different time intervals after administration of developed NPs compared with pure curcuminoids.

Table 9. Pharmacokinetic parameters of CUR-PNIPAM after i.n. and i.v. administration to rats at the dose of 0.100 mg kg⁻¹ in brain, lungs and plasma (n = 6, mean ± SD).

Formulation administration	Samples	Cmax (ng/mL g)	Tmax	t1/2 (h)	Ke (h^−1)	AUC0–t (ng min/mL g)
CUR (i.n.)	Brain	200.67 ± 14.90	2.00	21.04 ± 4.10	0.03294 ± 0.01	2476.17 ± 311.66
	Lungs	23.33 ± 3.20	2.00	23.36 ± 3.58	0.02967 ± 0.00472	276.33 ± 38.72
	Plasma	60.50 ± 4.32	0.5	6.43 ± 0.67	0.10784 ± 0.01141	450.63 ± 44.65
CUR (i.v.)	Brain	108.50 ± 13.02	2.00	7.48 ± 0.65	0.09268 ± 0.0082	1032.75 ± 67.16
	Lungs	177.67 ± 14.02	2.00	6.48 ± 1.00	0.10693 ± 0.0218	1570.88 ± 57.62
	Plasma	1314.17 ± 35.41	1.00	8.92 ± 0.81	0.07769 ± 0.0067	17894.96 ± 377.06
CUR-PNIPAM (i.n.)	Brain	989.33 ± 14.29	2.00	219.44 ± 33.64	0.00316 ± 0.00046	15597.13 ± 373.31
	Lungs	112.33 ± 8.78	2.00	82.51 ± 58.70	0.00840 ± 0.00419	1796.54 ± 148.20
	Plasma	419.50 ± 7.79	2.00	28.50 ± 0.77	0.02432 ± 0.00066	6424.79 ± 151.74
CUR-PNIPAM (i.v.)	Brain	410.83 ± 28.74	2.00	38.96 ± 14.50	0.01779 ± 0.01	6355.88 ± 262.95
	Lungs	215.33 ± 7.50	2.00	147.10 ± 1089.82	0.00471 ± 0.0044	3366.67 ± 142.22
	Plasma	1219.17 ± 23.21	1.00	9.27 ± 1.11	0.07480 ± 0.00893	17501.46 ± 297.96
CUR (i.n.)	Brain/plasma	3.32	4.00	3.27	0.31	5.49
CUR (i.v.)	Brain/plasma	0.08	2.00	0.84	1.19	0.06
CUR-PNIPAM (i.n.)	Brain/plasma	2.36	1.00	7.70	0.13	2.43
CUR-PNIPAM (i.v.)	Brain/plasma	0.34	2.00	4.20	0.14	0.36

Table 10. Pharmacokinetic parameters of DMC-PNIPAM after i.n. and i.v. administration to rats at the dose of 0.100 mg kg⁻¹ in brain, lungs and plasma (n = 6, mean ± SD).

Formulation administration	Samples	Cmax (ng/mL g)	Tmax	t1/2 (h)	Ke (h^−1)	AUC0–t (ng min/mL g)
DMC (i.n.)	Brain	205.50 ± 13.14	2.00	21.57 ± 3.44	0.03213 ± 0.00470	2560.92 ± 236.86
	Lungs	21.67 ± 4.41	2.00	19.31 ± 7.30	0.03590 ± 0.02245	240.13 ± 74.70
	Plasma	58.5 ± 6.53	0.50	5.85 ± 1.10	0.11855 ± 0.02614	411.29 ± 93.51
DMC (i.v.)	Brain	109.5 ± 22.37	2.00	7.37 ± 1.44	0.09405 ± 0.0093	1045.83 ± 72.00
	Lungs	176.83 ± 12.14	2.00	6.37 ± 2.94	0.10883 ± 0.0210	1551.96 ± 46.34
	Plasma	1297.50 ± 34.49	1.00	8.55 ± 2.16	0.08109 ± 0.0114	17503.29 ± 868.06
DMC-NIPAM (i.n.)	Brain	985.33 ± 18.42	2.00	195.11 ± 50.13	0.00355 ± 0.00091	15649.63 ± 782.57
	Lungs	113.83 ± 7.31	2.00	72.39 ± 61.76	0.00958 ± 0.00431	1814.79 ± 149.37
	Plasma	416.17 ± 9.13	2.00	27.89 ± 2.07	0.02485 ± 0.00206	6343.13 ± 119.57
DMC-NIPAM (i.v.)	Brain	411.17 ± 30.15	2.00	39.17 ± 12.22	0.01770 ± 0.00538	6377.17 ± 300.64
	Lungs	211.83 ± 4.96	2.00	74.55 ± 16.48	0.00930 ± 0.00233	3214.29 ± 99.68
	Plasma	1214.17 ± 11.60	1.00	9.45 ± 0.90	0.07337 ± 0.00671	17494.96 ± 299.53
DMC (i.n.)	Brain/plasma	3.51	4	3.69	0.27	6.23
DMC (i.v.)	Brain/plasma	0.08	2.00	0.86	1.16	0.06
DMC-NIPAM (i.n.)	Brain/plasma	2.37	1.00	7.00	0.14	2.47
DMC-NIPAM (i.v.)	Brain/plasma	0.34	2.00	4.14	0.24	0.36

Table 11. Pharmacokinetic parameters of BDMC-PNIPAM after i.n. and i.v. administration to rats at the dose of 0.100 mg kg⁻¹ in brain, lungs and plasma (n = 6, mean ± SD).

Formulation administration	Samples	Cmax (ng/mL g)	Tmax	t1/2 (h)	Ke (h^−1)	AUC0–t (ng min/mL g)
BDMC (i.n.)	Brain	202.17 ± 12.06	2.00	20.90 ± 3.84	0.03317 ± 0.00548	2493.38 ± 243.51
	Lungs	23.67 ± 5.09	2.00	20.94 ± 6.78	0.03310 ± 0.02244	269.17 ± 76.08
	Plasma	55.67 ± 6.31	0.50	5.94 ± 1.13	0.11661 ± 0.02664	367.00 ± 93.10
BDMC (i.v.)	Brain	105.83 ± 17.41	2.00	7.61 ± 1.69	0.09106 ± 0.0087	1002.13 ± 104.71
	Lungs	176.50 ± 12.88	2.00	6.32 ± 1.93	0.10964 ± 0.0207	1547.13 ± 50.36
	Plasma	1315.83 ± 54.52	1.00	8.89 ± 1.83	0.09106 ± 0.0069	17918.13 ± 375.04
BDMC-PNIPAM (i.n.)	Brain	989.83 ± 20.38	2.00	189.37 ± 53.32	0.00366 ± 0.00104	15668.92 ± 813.51
	Lungs	112.50 ± 7.56	2.00	70.37 ± 62.56	0.00985 ± 0.00432	1787.92 ± 145.87
	Plasma	414.67 ± 10.05	2.00	27.51 ± 2.14	0.02520 ± 0.00210	6309.33 ± 164.92
BDMC-PNIPAM (i.v.)	Brain	408.33 ± 31.61	2.00	41.25 ± 10.58	0.01680 ± 0.00390	6338.79 ± 312.59
	Lungs	209.50 ± 6.50	2.00	77.44 ± 17.84	0.00895 ± 0.00248	3192.96 ± 122.03
	Plasma	1218.33 ± 11.71	1.00	9.54 ± 0.87	0.07262 ± 0.00671	17569.75 ± 284.71
BDMC (i.n.)	Brain/plasma	3.63	4.00	3.52	0.28	6.79
BDMC (i.v.)	Brain/plasma	0.08	2.00	0.86	1.00	0.06
BDMC-PNIPAM (i.n.)	Brain/plasma	2.39	1.00	6.88	0.15	2.48
BDMC-PNIPAM (i.v.)	Brain/plasma	0.34	2.00	4.32	0.23	0.36

Table 12. Drug targeting efficiency and direct nose-to-brain transport following i.n. administration of different formulations.

			Comparative bioavailability^a (AUCi.n./AUCi.v) (%)
Formulations	Drug targeting efficiency (%DTE)^a	Direct nose-to-brain transport (%DTP)^a	Blood	Brain
CUR	203.97 ± 56.43	51.01 ± 8.09	12.53 ± 2.16	38.96 ± 5.36
CUR-PNIPAM	659.23 ± 83.59	84.03 ± 1.81	36.71 ± 5.61	245.40 ± 11.92
DMC	201.26 ± 53.79	52.62 ± 7.01	12.35 ± 2.63	40.16 ± 4.41
DMC-PNIPAM	667.84 ± 85.12	85.23 ± 2.19	36.26 ± 5.49	246.70 ± 12.65
BDMC	205.40 ± 54.76	53.64 ± 7.46	13.09 ± 2.77	39.34 ± 4.69
BDMC-PNIPAM	677.12 ± 289.99	85.47 ± 2.49	35.91 ± 5.31	247.19 ± 12.81

^aParameters are derived using mean ± standard error of mean values of six different estimations.

Figure 9. CUR-PNIPAM, DMC-PNIPAM and BDMC-PNIPAM pre-treatment for 21 days improves performances in the neurological deficits after stroke: (A) neurological scores and (B and C) quantification of TNF-α and IL-1β activity by ELISA in the sham, MCAO and CUR-PNIPAM, DMC-PNIPAM and BDMC-PNIPAM + MCAO groups. [***p < 0.001 (Sham vs MCAO); ##p < 0.01 and ###p < 0.001 (Treatment vs MCAO group)].

Figure 10. Representative photomicrographs showing the TS of rats' brain (A, B, C and D) and nasal mucosa (E, F, G and H) for normal control, PNIPAM-NPs (placebo), CUR-PNIPAM, DMC-PNIPAM and BDMC-PNIPAM-treated groups, respectively after 14 days.