Functionalized chitosan nanoparticles for cutaneous delivery of a skin whitening agent: an approach to clinically augment the therapeutic efficacy for melasma treatment

Figures & data

Table 1. $2^4$ Full factorial design setup showing the values of the dependent and independent factors.

Formula code	Concentration of different components (level of independent variables)				Dependent variables
	XA* (% w/v)	XB** (% w/v)	XC^† (% w/v)	XD^‡ (% w/v)	Mean P.S (nm) ± S.D.	Mean PDI ± S.D.	ζ-potential Mean ± S.D. (mV)	Mean EE% ± S.D.
C1	0.15	0.05	0.00	0.00	562.85 ± 17.22	0.51 ± 0.04	+41.65 ± 1.06	83.46 ± 4.94
C2	0.15	0.05	0.00	0.10	568.20 ± 24.55	0.54 ± 0.03	+40.85 ± 2.33	85.47 ± 0.33
C3	0.15	0.05	0.10	0.00	567.50 ± 13.44	0.53 ± 0.02	+39.95 ± 3.60	84.80 ± 3.89
C4	0.15	0.05	0.10	0.10	570.15 ± 18.17	0.55 ± 0.03	+38.90 ± 2.69	82.88 ± 5.97
C5	0.15	0.10	0.00	0.00	335.45 ± 14.78	0.33 ± 0.01	+38.18 ± 2.72	86.79 ± 3.78
C6	0.15	0.10	0.00	0.10	337.80 ± 10.18	0.32 ± 0.02	+37.85 ± 1.63	82.01 ± 4.19
C7	0.15	0.10	0.10	0.00	341.65 ± 29.20	0.37 ± 0.03	+36.55 ± 0.92	88.45 ± 2.96
C8	0.15	0.10	0.10	0.10	341.80 ± 0.28	0.34 ± 0.02	+37.01 ± 2.70	83.64 ± 4.26
C9	0.30	0.05	0.00	0.00	567.05 ± 12.79	0.54 ± 0.04	+41.15 ± 5.16	63.65 ± 4.04
C10	0.30	0.05	0.00	0.10	570.55 ± 11.80	0.56 ± 0.02	+38.95 ± 3.75	66.65 ± 3.18
C11	0.30	0.05	0.10	0.00	568.50 ± 7.77	0.60 ± 0.03	+39.00 ± 4.66	64.70 ± 5.87
C12	0.30	0.05	0.10	0.10	572.80 ± 28.57	0.55 ± 0.02	+38.23 ± 3.15	63.96 ± 4.65
C13	0.30	0.10	0.00	0.00	340.25 ± 28.64	0.33 ± 0.03	+41.71 ± 1.12	65.09 ± 4.08
C14	0.30	0.10	0.00	0.10	343.10 ± 20.65	0.36 ± 0.03	+38.01 ± 2.53	64.11 ± 5.57
C15	0.30	0.10	0.10	0.00	344.90 ± 7.21	0.35 ± 0.03	+37.90 ± 1.98	68.99 ± 3.89
C16	0.30	0.10	0.10	0.10	343.85 ± 21.00	0.38 ± 0.02	+37.80 ± 1.27	64.05 ± 5.06

*$X_A:$ concentration of chitosan; **$X_B:$ concentration of TPP; ^†$X_C:$ concentration of HA; ^‡$X_D:$ concentration of collagen.

Figure 1. Three-dimensional surface plot of the influence of chitosan concentration and TPP concentration on: (a) P.S of the prepared CSNPs in the absence and presence of HA and collagen, (b) PDI of the prepared CSNPs in the absence and presence of HA and collagen, (c) EE% of α-arbutin in the prepared CSNPs in absence and presence of HA and collagen.

Table 2. Checkpoint analysis for P.S, PDI and EE% models for the evaluation of the adequacy of the developed models.

XA*	XB**	XC^†	XD^‡	Actual value ±S.D.	Predicted value	Bias (%)
P.S (nm) model
0.20	0.08	0.00	0.00	473.20 ± 11.031	427.93	9.72
0.20	0.08	0.00	0.10	478.25 ± 10.25	431.33	9.81
0.20	0.08	0.10	0.00	475.95 ± 18.31	432.77	9.07
0.20	0.08	0.10	0.10	482.55 ± 17.61	433.90	10.10
PDI model
0.20	0.08	0.00	0.00	0.411 ± 0.007	0.406	1.09
0.20	0.08	0.00	0.10	0.418 ± 0.005	0.420	0.48
0.20	0.08	0.10	0.00	0.412 ± 0.009	0.440	6.44
0.20	0.08	0.10	0.10	0.404 ± 0.013	0.430	7.06
Entrapment efficiency (%) model
0.20	0.08	0.00	0.00	78.06% ± 8.77	78.47%	0.53
0.20	0.08	0.00	0.10	75.55% ± 6.96	80.75%	6.88
0.20	0.08	0.10	0.00	77.17% ± 8.50	82.72%	7.21
0.20	0.08	0.10	0.10	76.66% ± 5.30	76.89%	0.31

*$X_A:$ concentration of chitosan; **$X_B:$ concentration of TPP; ^†$X_C:$ concentration of HA; ^‡$X_D:$ concentration of collagen.

Table 3. Release kinetics and parameters of α-arbutin from the selected chitosan nanoparticles; dispersions and hydrogels versus their controls.

Formula	Correlation coefficient (R^2) (CSNPs dispersions)			Korsmeyer–Peppas (n values for release mechanism confirmation)	T50 (h) Mean ± S.D.	T90 (h) Mean ± S.D.	Correlation coefficient (R^2) (CSNPs hydrogels)			Korsmeyer–Peppas (n values for release mechanism confirmation)	T50 (h) Mean ± S.D.	T90 (h) Mean ± S.D.
	Zero order	First order	Higuchi model				Zero order	First order	Higuchi model
C5	0.732	0.986	0.985	0.430	2.55 ± 0.04	7.96 ± 0.04	0.758	0.976	0.988	0.50	4.41 ± 0.11	11.09 ± 0.14
C6	0.781	0.989	0.992	0.452	3.15 ± 3.54	8.24 ± 0.18	0.751	0.979	0.995	0.50	4.39 ± 0.13	11.01 ± 0.12
C7	0.846	0.989	0.985	0.395	4.41 ± 0.11	8.23 ± 0.65	0.842	0.996	0.998	0.43	5.01 ± 0.20	11.59 ± 0.05
C8	0.772	0.988	0.987	0.363	3.47 ± 0.21	9.47 ± 0.77	0.800	0.987	0.989	0.42	4.83 ± 0.15	11.12 ± 0.12
Control	0.447	0.982	0.764	0.12	1.15 ± 0.13	2.31 ± 0.22	0.515	0.988	0.822	0.14	2.23 ± 0.10	3.56 ± 0.11

Figure 2. Release profile of α-arbutin in PBS (pH 7.4) from: (a) CSNPs dispersions compared to drug solution and (b) CSNPs hydrogels compared to the control drug hydrogel.

Figure 3. Percent α-arbutin ‘deposited in/permeated through’ skin layers from the selected formulations (C5–C8) as: (a) liquid dispersions compared to drug solution in PBS (pH, 7.4) and (b) gels compared to drug hydrogel control.

Figure 4. TEM photomicrographs of selected α-arbutin loaded CSNPs (C8) at 30000× (a), and 8000× (b).

Figure 5. Two female patients with epidermal melasma: (A and C) photos for both sides of the face; right and lift, respectively before treatment showing brown spotty patches along the malar region, (B) the right side of the face after 2 months of treatment showing mild improvement in group I after topical application of C5 hydrogel (a) and good improvement in group II after topical application of C8 hydrogel (b), (D) the left side of the face after 2 months of treatment showing no improvement in both females after topical application of α-arbutin hydrogel.

Table 4. Comparison between groups I treated with CSNPs-C5 gel and group II treated with CSNPs-C8 gel versus α-arbutin hydrogel (as control) with regards to the percent reduction of the clinical and histopathologic parameters; mMASI scores, MPSA and MART-1-positive cells number (n = 10 patients).

Group	Mean reduction (%) of mMASI scores^‡		Mean reduction (%) of MPSA^ϕ (μm^2)		MART-1-positive cells number^#
	Right side of face α-arbutin-CSNPs hydrogels	Left side of face α-arbutin hydrogels (control)	Right side of face α-arbutin-CSNPs hydrogels	Left side of face α-arbutin hydrogels (control)	Right side of face α-arbutin-CSNPs hydrogels	Left side of face α-arbutin hydrogels (control)
I (treated with CSNPs-C5* gel)	24.6 ± 6.8 (Mild; 1%-25%)	4.3 ± 2.7 (Mild; 1%-25%)	31.3 ± 3.8	1.7 ± 1.3	40.6 ± 4.5	10.6 ± 7.9
II (treated with CSNPs- C8** gel)	41.2 ± 6.5 (Moderate; 26%-50%)	3.1 ± 2.5 (Mild; 1%-25%)	37.8 ± 5.1	1.96 ± 0.98	47.5 ± 18.5	9.4 ± 6.7

^‡Modified Melasma Area and Severity Index; ^Φmelanin particle surface area; ^#monoclonal mouse anti–melanoma antigen recognized by T cells-1.

*C5 formulated with: 0.15% chitosan, 0.1 % TPP, 0.0% HA and 0.0% collagen; **C8 formulated with: 0.15% chitosan; 0.1 % TPP, 0.1% HA and 0.1% collagen.

Figure 6. Two groups comprising skin biopsies from epidermal melasma lesions before and after treatment: a) Group I and b) Group II. The untreated right and left sides are denoted by E and G respectively, showing increased density of melanin in the basal layer of the epidermis. Biopsies from the right side of the face after 2 months of treatment (F) demonstrated significant decrease in epidermal MPSA in groups I and II after the application of C5 and C8 hydrogels, respectively. Biopsies from the left side of the face after 2 months of treatment (H) demonstrated insignificant decrease in epidermal MPSA in both groups after topical application of α-arbutin hydrogel (MF stain X200).

Figure 7. Two groups comprising skin biopsies from epidermal melasma lesions before and after treatment (a) Group I and (b) Group II. The untreated right and left sides are denoted by I and K respectively, showing increased number of MART-1-positive stained cells in the basal layer of the epidermis. After 2 months of treatment with the nano-formulations gels, (J) biopsies demonstrated significant decrease in the number of epidermal MART-1-positive cells in groups I and II after topical application of C5 and C8 hydrogels respectively. Biopsies from the left side of the face after 2 months of treatment with α-arbutin hydrogel (L) demonstrated insignificant decrease in the number of epidermal MART-1-positive cells in both groups (MART-1 stain X200).