PEGylated Tween 80-functionalized chitosan-lipidic nano-vesicular hybrids for heightening nose-to-brain delivery and bioavailability of metoclopramide

Figures & data

Table 1. Draper-Lin small composite design (DLD) independent and dependent variables with the chosen responses and assigned goals of MTC–loaded lipidic nano-vesicular hybrids.

Independent variables	Low	Medium	High	Units
X1: Chitosan amount	0	25	50	mg
X2: Cholesterol conc.	0.5	0.75	1	Molar ratio
X3: PEG 600 conc.	0	15	30	%
X4: Tween 80 conc.	0	15	30	%
Dependent variables	Units	Goals
Y1: Mean vesicle size	nm	Minimize
Y2: Zeta potential	mV	Maximize
Y3: Entrapment efficiency	%	Maximize
Y4: Initial release	%	Maximize/ Minimize
Y5: Cumulative release	%	Maximize/ Minimize

Table 2. Experimental runs and observed values of responses for DLD MTC–loaded lipidic nano-vesicular hybrids, along with the two optimized formulations (Opti-Max and Opti-Min MTC–loaded PEG-T-Chito-Lip nano-vesicular formulations).^a

Runs (#)	Factors				Responses
	X1	X2	X3	X4	Y1		Y2		Y3		Y4		Y5
	(mg)	(Molar ratio)	(%)	(%)	(nm)	± SD	(mV)	± SD	(%)	± SD	(%)	± SD	(%)	± SD
F1	0	0.5	0	0	212.60	2.60	−27.3	0.65	59.60	0.86	27.3	3.6	95.7	4.3
F2	50	0.5	0	30	392.84	1.33	41.2	0.89	91.33	2.73	18.8	0.9	81.5	0.6
F3	50	1	0	0	456.67	2.69	45.9	0.50	89.45	1.38	3.4	0.7	68.8	0.9
F4	25	0.75	15	0	357.23	1.51	41.5	0.67	79.36	1.59	9.4	0.8	71.9	3.8
F5	50	1	30	0	517.33	3.96	37.8	0.68	89.95	1.60	2.6	0.4	51.9	1.6
F6	25	1	15	15	378.27	0.98	40.4	0.95	84.67	0.80	5.3	0.6	74.4	4.1
F7	25	0.75	30	15	455.43	3.50	35.6	0.98	86.21	2.80	10.2	1.5	82.7	2.7
F8	25	0.75	0	15	336.10	1.60	47.8	1.05	77.93	0.33	15.9	0.7	89.5	2.4
F9	25	0.5	15	15	340.95	0.85	46.7	0.97	82.58	2.54	19.4	0.5	91.7	0.8
F10	50	0.75	15	15	437.80	0.57	39.9	0.45	90.39	3.56	9.3	1.6	75.6	1.7
F11	0	1	30	30	387.31	2.83	−35.9	0.63	74.15	1.73	12.3	0.8	83.1	2.2
F12	50	0.5	30	30	451.46	1.33	30.1	0.88	93.85	3.35	8.9	0.2	72.4	2.9
F13	0	0.5	30	0	297.17	2.64	−32.4	0.64	66.30	1.46	16.5	1.2	87.6	1.7
F14	0	1	0	30	321.36	1.42	−29.5	0.95	71.30	2.24	10.8	1.9	82.9	3.8
F15	25	0.75	15	30	377.40	2.08	38.2	1.67	85.13	1.80	15.4	1.1	87.3	3.4
F16	0	0.75	15	15	283.20	0.64	−34.6	0.94	69.18	3.60	16.6	1.7	89.1	4.1
F17	25	0.75	15	15	326.53	1.06	40.3	0.49	85.35	4.30	12.2	0.9	86.4	2.4
F18	25	0.75	15	15	304.68	1.82	41.4	0.86	88.42	2.25	17.9	0.8	88.7	3.8
F19	25	0.75	15	15	349.30	0.95	38.9	0.48	82.68	2.90	15.6	0.5	91.8	2.7
Opti-Max	26.5	0.5	0.9	22	344.90	1.84	39.7	2.33	79.64	0.89	21.9	2.7	95.6	3.9
Opti-Min	31.8	1	10.7	0.05	405.67	3.67	34.8	3.15	86.45	2.91	5.3	0.6	64.2	4.3

^a All formulations were formulated with 1 molar ratio Span 60. X₁ is the chitosan amount (mg), X₂ is the cholesterol molar ratio concentration, X₃ is the PEG 600 concentration percentage, X₄ is the Tween 80 concentration percentage, Y₁ is the mean vesicle size (nm), Y₂ is the zeta potential (mV), Y₃ is the entrapment efficiency percentage, Y₄ is the percentage of MTC initial release after 1 h and Y₅ is the percentage of MTC cumulative release after 24 h.

Figure 1. Schematic illustration of the performed valuation of nose-to-brain delivery and in-vivo pharmacokinetics of the intranasal dual-optimized MTC–loaded PEG-T-Chito-Lip nano-vesicular ISG. Abbreviations: MTC, metoclopramide hydrochloride; PEG-T-Chito-Lip nano-vesicular hybrid, PEGylated Tween 80–functionalized chitosan–lipidic nano-vesicular hybrid; ISG, in-situ gel; I.V., intravenous.

Figure 2. In-vitro release profile of MTC from different DLD formulations, Opti-Max and Opti-Min MTC–loaded PEG-T-Chito-Lip nano-vesicular formulations. Abbreviations: MTC, metoclopramide hydrochloride; PEG-T-Chito-Lip nano-vesicular hybrid, PEGylated Tween 80–functionalized chitosan–lipidic nano-vesicular hybrid.

Table 3. Kinetics studies of the release profiles of MTC from different DLD formulations, as well as the two optimized formulations (Opti-Max and Opti-Min MTC–loaded PEG-T-Chito-Lip nano-vesicular formulations).

Formulations	(r) value				Korsmeyer-Peppas model (n) value^a	Release Rate constant
	Zero order	First order	Second order	Higuchi Diffusion model
F1	0.935321	–0.99731	0.949483	0.978033	0.44475137	−0.127630
F2	0.986926	–0.99818	0.973069	0.996403	0.46061692	−0.063720
F3	0.898888	−0.93767	0.961799	0.961944	0.99187883	18.90613
F4	0.866456	−0.90640	0.934575	0.942258	0.66274254	17.47046
F5	0.838950	−0.88444	0.921519	0.933285	0.92237558	13.11036
F6	0.836004	−0.88437	0.918715	0.924240	0.88219142	19.85236
F7	0.955236	–0.99835	0.983468	0.995524	0.63954735	−0.069830
F8	0.917845	–0.98835	0.987232	0.977366	0.57221493	−0.09258
F9	0.876106	−0.97415	0.993392	0.955028	0.52374740	0.004853
F10	0.899956	−0.96526	0.996377	0.968950	0.66510038	0.001315
F11	0.893591	−0.94247	0.959881	0.960843	0.63278352	19.72367
F12	0.857584	−0.90648	0.935262	0.940096	0.70925395	18.09740
F13	0.879566	−0.93564	0.952126	0.953743	0.56301198	19.94656
F14	0.897427	−0.93762	0.949219	0.960782	0.64463406	19.61249
F15	0.894940	−0.93751	0.948753	0.959247	0.58513901	20.34569
F16	0.873153	−0.93411	0.949304	0.950573	0.56227996	20.16422
F17	0.912531	−0.9848	0.994240	0.974829	0.63187375	0.002762
F18	0.896471	−0.98079	0.994967	0.966964	0.53027348	0.003401
F19	0.896336	−0.98072	0.983412	0.964444	0.58287113	0.004922
Opti-Max	0.893369	–0.97772	0.977415	0.960530	0.51874445	−0.13430
Opti-Min	0.819541	−0.87222	0.915203	0.917608	0.84136923	16.60406

^a n ≤ 0.5 indicates Case I transport; Fickian diffusion; Higuchi release; t½ dependence, while n = 0.5-1 indicates Non-Fickian release; anomalous, the release is controlled by a combination of diffusion and polymer relaxation.

Table 4. Estimated effects of factors and associated p-values for responses.

Factors	Y1		Y2		Y3		Y4		Y5
	Factor effect	P value	Factor effect	P value	Factor effect	P value	Factor effect	P value	Factor effect	P value
X1	154.60	0.037^a	5.30	0.366	21.21	0.009^a	−7.30	0.121	−13.50	0.059
X2	37.32	0.498	−6.30	0.293	2.09	0.665	−14.10	0.019^a	−17.30	0.028^a
X3	77.83	0.026^a	−3.98	0.163	4.17	0.106	−5.14	0.037^a	−8.14	0.024^a
X4	20.18	0.708	−3.30	0.561	5.77	0.268	6	0.182	15.40	0.040^a
X1^2	−4.72	0.921	−9.84	0.100	−6.59	0.173	−0.16	0.963	−5.16	0.322
X1X2	2.87	0.962	−1.63	0.794	−0.56	0.916	5.75	0.239	11.43	0.118
X1X3	−7.81	0.771	−7.68	0.042^a	−1.63	0.507	−0.35	0.860	−4.53	0.153
X1X4	−44.83	0.469	−10.83	0.137	−1.35	0.801	−3.50	0.448	−4.68	0.462
X2^2	−6.50	0.891	2.76	0.583	1.09	0.797	−1.36	0.701	−3.76	0.456
X2X3	−4.14	0.877	1.08	0.701	−1.47	0.548	5.35	0.045^a	0.13	0.964
X2X4	4.63	0.938	−2.18	0.728	−2.09	0.698	1	0.822	5.18	0.419
X3^2	65.80	0.214	−0.94	0.848	−2.01	0.640	0.038	0.991	2.34	0.636
X3X4	−5.16	0.847	−0.43	0.878	−0.46	0.849	0.80	0.689	4.025	0.193
X4^2	8.90	0.851	−4.64	0.372	−1.67	0.697	−1.26	0.722	−10.66	0.080

X₁ is the chitosan amount (mg), X₂ is the cholesterol molar ratio concentration, X₃ is the PEG 600 concentration percentage, X₄ is the Tween 80 concentration percentage, X₁X₂, X₁X₃, X₁X₄, X₂X₃, X₂X₄, X₃X₄ are the interaction terms between the factors, X₁², X₂², X₃², X₄² are the quadratic terms of the factors, Y₁ is the mean vesicle size (nm), Y₂ is the zeta potential (mV), Y₃ is the entrapment efficiency percentage, Y₄ is the percentage of MTC initial release after 1 h and Y₅ is the percentage of MTC cumulative release after 24 h.

^a Significant effect of factors on individual responses.

Figure 3. Pareto chart showing the standardized effects of factors on the observed responses (Y₁–Y₅).

Figure 4. Contour-response surface plot showing the effects of factors on the observed responses (Y₁–Y₅).

Figure 5. Light (left figures) and TEM (right figures) micrographs of the Opti-Max (A and B) and Opti-Min (C and D) MTC–loaded PEG-T-Chito-Lip nano-vesicular formulations. Abbreviations: MTC, metoclopramide hydrochloride; PEG-T-Chito-Lip nano-vesicular hybrid, PEGylated Tween 80–functionalized chitosan–lipidic nano-vesicular hybrid.

Figure 6. DSC/TGA thermograms of the pure MTC, Opti-Min and Opti-Max MTC–loaded PEG-T-Chito-Lip nano-vesicular formulations. Abbreviations: MTC, metoclopramide hydrochloride; PEG-T-Chito-Lip nano-vesicular hybrid, PEGylated Tween 80–functionalized chitosan–lipidic nano-vesicular hybrid.

Figure 7. Plasma, brain and liver MTC concentration vs time profiles after administration of the intranasal dual-optimized MTC–loaded PEG-T-Chito-Lip nano-vesicular ISG, intranasal raw MTC-loaded ISG, MTC commercial oral tablet, and MTC commercial I.V. injection, in Sprague Dawley rats (n = 3). The dose of MTC in all formulations was equivalent to 3.5 mg/kg body weight. *, #, and $indicate P < 0.05 versus intranasal raw MTC-loaded ISG, MTC commercial oral tablet, and MTC commercial I.V. injection, respectively. Abbreviations: MTC, metoclopramide hydrochloride; PEG-T-Chito-Lip nano-vesicular hybrid, PEGylated Tween 80–functionalized chitosan–lipidic nano-vesicular hybrid; ISG, in-situ gel; I.V., intravenous.

Table 5. The PK parameters of MTC in the plasma, brain and liver after administration of the intranasal dual-optimized MTC–loaded PEG-T-Chito-Lip nano-vesicular ISG, intranasal raw MTC-loaded ISG, MTC commercial oral tablet, and MTC commercial I.V. injection, in Sprague Dawley rats (n = 3).

PK parameters of MTC in the plasma	Intranasal dual-optimized MTC–loaded nano-vesicular ISG		Intranasal raw MTC-loaded ISG		MTC commercial oral tablet		MTC commercial I.V. injection
	Value	(± SD)	Value	(± SD)	Value	(± SD)	Value	(± SD)
${\mathrm{K}}_{\mathrm{el}}\mathrm{ }\left({\mathrm{h}}^{-1}\right)$	0.44	0.012	0.41	0.16	0.62	0.21	0.29	0.15
${\mathrm{t}}_{\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$2$}\right.}\mathrm{ }\left(\mathrm{h}\right)$	1.58	0.045	1.86	0.62	1.19	0.34	2.81	1.18
${\mathrm{T}}_{\max }\mathrm{ }\left(\mathrm{h}\right)$	1	NA	1	NA	1	NA	–	–
${\mathrm{C}}_{\max }\mathrm{ }\left(\mathrm{ng}/\mathrm{ml}\right)$	1375.33^a^, b	61.58	672.33^b, c	69.21	450^c	38.43	1193	79.57
${\mathrm{AUC}}_{0\text{-}\mathrm{t}}\mathrm{ }\left(\mathrm{ng}/\mathrm{ml}\times \mathrm{h}\right)$	3237.17^a^, b, c	220.84	1907.42^b, c	94.49	1174^c	82.64	2340.58	66.69
${\mathrm{AUC}}_{0\text{-}\mathrm{\infty }}\mathrm{ }\left(\mathrm{ng}/\mathrm{ml}\times \mathrm{h}\right)$	3765.06^a, b, c	294.98	2320.11^b	359.79	1278.91^c	136.27	2949.92	333.16
${\mathrm{MRT}}_{0\text{-}\mathrm{\infty }}\mathrm{ }\left(\mathrm{h}\right)$	2.68	0.1	3.05	0.69	2.36	0.34	3.18	0.91
${\mathrm{V}}_{\mathrm{d}}\mathrm{ }\left[\left(\mathrm{mg}/\mathrm{kg}\right)/\left(\mathrm{ng}/\mathrm{ml}\right)\right]$	2 х10^–3 a, b	0.1 х10^–3	3.98 х10^–3	0.8 х10^–3	4.7 х10^–3	1.2 х10^–3	4.7 х10^–3	1.62 х10^–3
$\mathrm{Cl}\mathrm{ }\left(\raisebox{1ex}{$\left[\left(\mathrm{mg}/\mathrm{kg}\right)/\left(\mathrm{ng}/\mathrm{ml}\right)\right]$}\!\left/ \!\raisebox{-1ex}{$\mathrm{h}$}\right.\right)$	9.3 х10^–4 a, b, c	0.7 х10^–4	15.3 х10^–4 b	2.4 х10^–4	2.8 х10^–3 c	0.3 х10^–3	1.2 х10^–3	1.4 х10^–4
PK parameters of MTC in the brain	Value	(± SD)	Value	(± SD)	Value	(± SD)	Value	(± SD)
${\mathrm{K}}_{\mathrm{el}}\mathrm{ }\left({\mathrm{h}}^{-1}\right)$	0.38^c	0.08	0.60^b	0.12	0.33^c	0.01	0.70	0.15
${\mathrm{t}}_{\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$2$}\right.}\mathrm{ }\left(\mathrm{h}\right)$	1.86^c	0.37	1.18^b	0.21	2.10^c	0.04	1.01	0.21
${\mathrm{T}}_{\max }\mathrm{ }\left(\mathrm{h}\right)$	1	NA	1	NA	1	NA	0.83	0.29
${\mathrm{C}}_{\max }\mathrm{ }\left(\mathrm{ng}/\mathrm{g}\right)$	1856.33^a, b, c	86.09	1366.33^b, c	97.34	333.67^c	42.55	507.00	37.04
${\mathrm{AUC}}_{0\text{-}\mathrm{t}}\mathrm{ }\left(\mathrm{ng}/\mathrm{g}\times \mathrm{h}\right)$	6416.92^a, b, c	205.14	2878.25^b, c	121.13	666.58^c	88.01	1149.08	84.25
${\mathrm{AUC}}_{0\text{-}\mathrm{\infty }}\mathrm{ }\left(\mathrm{ng}/\mathrm{g}\times \mathrm{h}\right)$	8206.36^a, b, c	336.74	3109.06^b, c	217.29	750.13^c	92.46	1215.73	106.79
${\mathrm{AUC}}_{0\text{-}\mathrm{t}/0\text{-}\mathrm{\infty }}$	0.78^a, b, c	0.05	0.93	0.03	0.89^c	0.01	0.95	0.02
${\mathrm{MRT}}_{0\text{-}\mathrm{\infty }}\mathrm{ }\left(\mathrm{h}\right)$	3.47^a, b, c	0.40	2.19	0.28	2.41^c	0.10	1.99	0.20
${\mathrm{V}}_{\mathrm{d}}\mathrm{ }\left[\left(\mathrm{mg}/\mathrm{kg}\right)/\left(\mathrm{ng}/\mathrm{g}\right)\right]$	1.1 х10^–3 a, b, c	0.2 х10^–3	1.9 х10^–3 b, c	0.2 х10^–3	0.014^c	2.02 х10^–3	4.2 х10^–3	0.7 х10^–3
$\mathrm{Cl}\mathrm{ }\left(\raisebox{1ex}{$\left[\left(\mathrm{mg}/\mathrm{kg}\right)/\left(\mathrm{ng}/\mathrm{g}\right)\right]$}\!\left/ \!\raisebox{-1ex}{$\mathrm{h}$}\right.\right)$	0.4 х10^–3 a, b, c	0.2 х10^–4	1.1 х10^–3 b, c	0.1 х10^–3	0.01^c	0.1 х10^–2	0.3 х10^–2	0.3 х10^–3
The PK parameters of MTC in the liver	Value	(± SD)	Value	(± SD)	Value	(± SD)	Value	(± SD)
${\mathrm{K}}_{\mathrm{el}}\mathrm{ }\left({\mathrm{h}}^{-1}\right)$	0.3	0.12	0.34	0.05	0.44	0.08	0.41	0.17
${\mathrm{t}}_{\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$2$}\right.}\mathrm{ }\left(\mathrm{h}\right)$	2.55	0.83	2.08	0.36	1.60	0.31	1.87	0.68
${\mathrm{T}}_{\max }\mathrm{ }\left(\mathrm{h}\right)$	2	NA	2	NA	1	NA	1	NA
${\mathrm{C}}_{\max }\mathrm{ }\left(\mathrm{ng}/\mathrm{g}\right)$	181.33^a, b, c	16.07	418.00^b, c	68.51	1322.67^c	47.09	571.00	62.23
${\mathrm{AUC}}_{0\text{-}\mathrm{t}}\mathrm{ }\left(\mathrm{ng}/\mathrm{g}\times \mathrm{h}\right)$	559.58^a, b, c	78.09	1295.75^b, c	224.56	4613.50^c	219.08	1891.75	111.98
${\mathrm{AUC}}_{0\text{-}\mathrm{\infty }}\mathrm{ }\left(\mathrm{ng}/\mathrm{g}\times \mathrm{h}\right)$	862.19^a, b, c	252.33	1799.69^b	451.03	5732.17^c	146.24	2430.83	310.64
${\mathrm{MRT}}_{0\text{-}\mathrm{\infty }}\mathrm{ }\left(\mathrm{h}\right)$	4.78	1.24	4.07	0.51	3.38	0.39	3.53	0.83
${\mathrm{V}}_{\mathrm{d}}\mathrm{ }\left[\left(\mathrm{mg}/\mathrm{kg}\right)/\left(\mathrm{ng}/\mathrm{g}\right)\right]$	1.5 х10^–2 a, b, c	0.3 х10^–2	0.6 х10^–2 b, c	0.1 х10^–2	0.1 х10^–2 c	0.2 х10^–3	0.4 х10^–2	0.1 х10^–2
$\mathrm{Cl}\mathrm{ }\left(\raisebox{1ex}{$\left[\left(\mathrm{mg}/\mathrm{kg}\right)/\left(\mathrm{ng}/\mathrm{g}\right)\right]$}\!\left/ \!\raisebox{-1ex}{$\mathrm{h}$}\right.\right)$	0.4 х10^–2 a, b, c	0.1 х10^–2	0.2 х10^–2 b	0.5 х10^–3	0.1 х10^–2 c	0.2 х10^–4	0.1 х10^–2	0.2 х10^–3

The dose of MTC in all formulations was equivalent to 3.5 mg/kg body weight.

^a Significant difference from values of intranasal raw MTC-loaded ISG with p-value <0.05.

^b Significant difference from values of o MTC commercial oral tablet with p-value <0.05.

^c Significant difference from values of o MTC commercial I.V. injection with p-value <0.05.

NA; not applicable.

Table 6. The absolute and relative bioavailability parameters of MTC in the plasma after administration of the intranasal dual-optimized MTC–loaded PEG-T-Chito-Lip nano-vesicular ISG and intranasal raw MTC-loaded ISG in Sprague Dawley rats (n = 3).

Parameters (unit)	Intranasal dual-optimized MTC–loaded nano-vesicular ISG		Intranasal raw MTC-loaded ISG		MTC commercial oral tablet
	Value	(± SD)	Value	(± SD)	Value	(± SD)
${\mathrm{F}}_{\mathrm{absolute}}\mathrm{ }(\mathrm{\%})$	128.42^a, b	13.44	78.49^b	5.48	43.5	3.72
${\mathrm{F}}_{\mathrm{relative}-\text{oral MTC tablet}}\mathrm{ }(\mathrm{\%})$	295.02^a	10.84	180.92	12.66	–	–
${\mathrm{F}}_{\mathrm{relative}-\text{intranasal pure MTC}}\mathrm{ }(\mathrm{\%})$	163.9	17.93	–	–	–	–

The dose of MTC in all formulations was equivalent to 3.5 mg/kg body weight. MTC commercial I.V. injection and MTC commercial oral tablet were set as references for the calculation of the absolute and relative bioavailability, respectively.

^a Significant difference from values of intranasal raw MTC-loaded ISG with p-value <0.05.

^b Significant difference from values of o MTC commercial oral tablet with p-value <0.05.

Figure 8. Evaluation of the nose-to-brain delivery of the dual-optimized MTC–loaded PEG-T-Chito-Lip nano-vesicular ISG formulation. a; MTC Brain/Blood ratio vs time of the intranasal dual-optimized MTC–loaded PEG-T-Chito-Lip nano-vesicular ISG, intranasal raw MTC-loaded ISG, MTC commercial oral tablet, and MTC commercial I.V. injection, in Sprague Dawley rats (n = 3). b; DTE%, DTP%, AUC_bf and DTI of the intranasal dual-optimized MTC–loaded PEG-T-Chito-Lip nano-vesicular ISG and intranasal raw MTC-loaded ISG relative to the MTC commercial I.V. injection, in Sprague Dawley rats (n = 3). The dose of MTC in all formulations was equivalent to 3.5 mg/kg body weight. *, #, and $indicate p < 0.05 versus intranasal raw MTC-loaded ISG, MTC commercial oral tablet, and MTC commercial I.V. injection, respectively. Abbreviations: MTC, metoclopramide hydrochloride; PEG-T-Chito-Lip nano-vesicular hybrid, PEGylated Tween 80–functionalized chitosan–lipidic nano-vesicular hybrid; ISG, in-situ gel; I.V., intravenous; DTE: drug targeting efficiency; AUC_bf: the AUC of the brain fraction; DTP: nose-to-brain direct transport percentage; DTI: drug targeting index.

Figure 9. Histological images of the rat nasal mucosa after 5 h treatment with the intranasal dual-optimized MTC–loaded PEG-T-Chito-Lip nano-vesicular ISG (a) and intranasal raw MTC-loaded ISG (b), along with the normal untreated rat nasal mucosa (c) at time zero. The nasal cavity in all images showed apparently normal nasal mucosa (H&E).