Inclusion of IR-820 into Soybean-Phosphatides-Based Nanoparticles for Near-Infrared-Triggered Release and Endolysosomal Escape in HaCaT Keratinocytes at Insignificant Cytotoxic Level

Figures & data

Table 1 Nanoparticle Formulations with the Hydrating Medium Volume of 10 mL

Formulation Code	NP	PEm-NP	PEm-IR-NP	DMA-NP	DMA-IR-NP	IR-NP	BCIP-IR-NP	TRITC-Dex40-NP	TRITC-Dex40-IR-NP
Organic phase (solvent: chloroform-methanol (2:1 v/v) mixture)
Soybean phosphatides (mg)	100	100	100	100	100	100	100	100	100
Cholesterol (mg)	7.4	7.4	7.4	7.4	7.4	7.4	7.4	7.4	7.4
Sodium deoxycholate (mg)	17.6	17.6	17.6	17.6	17.6	17.6	17.6	17.6	17.6
IR-820 (μg)	–	–	85	–	85	85	85	–	85
DMA (μg)	–	–	–	206.3	206.3	–	–	–	–
P. emblica fruit extract (PEm, mg GAE)	–	3.2	3.2	–	–	–	–	–	–
Aqueous phase (hydrating medium solvent: 1×PBS pH 7.4)
P. emblica fruit extract (PEm, mg GAE)	–	3.2	3.2	–	–	–	–	–	–
BCIP (mg)	–	–	–	–	–	–	50	–	–
TRITC-Dex40 (mg)	–	–	–	–	–	–	–	12.5	12.5

Abbreviations: PEm, Phyllanthus emblica L. fruit extract; IR, IR-820; DMA, 9,10-dimethylanthracene; BCIP, 5-bromo-4-chloro-3-indolyl phosphate disodium salt; TRITC-Dex40, tetramethylrhodamine isothiocyanate-dextran (molecular weight = 40kDa); GAE, gallic acid equivalent.

Figure 1 Absorption spectra of (A) DMA-NP, (B) DMA-IR-NP, (C) Absorbance ratio of DMA-IR-NP to DMA-NP at the peak wavelengths of 380 and 402 nm, (D) Fluorescence spectra of DMA-IR-NP setting the excitation wavelength at 375 nm, (E) Fluorescence intensity ratio of DMA-IR-NP to DMA-NP at the peak emission wavelength of 429 nm after being exposed to 830-nm NIR at 0.0118 W/cm² at different time points, and (F) Explanatory illustration of the scenario implied by (C, E).

Abbreviations: DMA, 9,10-dimethylanthracene; IR, IR-820; ³O₂, triplet oxygen; ¹O₂, singlet oxygen; NIR, near infrared.

Figure 2 Negative-stained TEM images of (A) NP, (B) PEm-NP, (C) PEm-IR-NP, (D) corresponding intensity-weighted hydrodynamic size distribution, (E) correlograms, and (F) absorption spectra. The scale bars are displayed on each image.

Abbreviations: TEM, transmission electron microscope; PEm, Phyllanthus emblica L. fruit extract; IR, IR-820.

Figure 3 (A) Negative-stained TEM images of PEm-IR-NP irradiated at different fluences of 830-nm NIR, (B) A schematic illustration representing the consequences on PEm-IR-NP corresponding to the NIR fluence, and (C) The increases in the lipid hydroperoxides in PEm-IR-NP (phospholipids concentration = 4 mg/mL) irradiated with various fluences of 830-nm NIR when normalized with the concentration of the lipid hydroperoxides in PEm-IR-NP before irradiation. Different letters above the box plots indicate the statistically significant differences (P < 0.001) and n = 8. Scale bar = 100 nm.

Abbreviations: TEM, transmission electron microscope; PEm, Phyllanthus emblica L. fruit extract; IR, IR-820; NIR, near infrared.

Figure 4 The release profile of the TPC from PEm-IR-NP into the stirred simulated sweat solution (pH 5.4) maintained at 32°C with and without 830-nm irradiation at 0.0118 W/cm² for the first 30 minutes.

Abbreviations: TPC, total phenolic content; PEm, Phyllanthus emblica L. fruit extract; IR, IR-820; M_t, the total amount of diffusing the TPC at time t; M_∞, the total amount of diffusing the TPC after infinite time.

Table 2 Characterization of the Release Profile Curves of TPCs from PEm-IR-NP into the Stirred Simulated Sweat Solution (pH 5.4) Maintained at 32°C with and without 830-nm Irradiation at 0.0118 W/cm² for the First 30 Minutes

Irradiation	t25% (hr)	t50% (hr)	t75% (hr)	MDT (hr)	DE2hr (%)
(-)	0.161	2.990	10.220	3.587	36.49
(+)	0.036	0.183	1.653	0.196	68.59

Abbreviations: TPC, total phenolic content; PEm, Phyllanthus emblica L. fruit extract; IR, IR-820; t_25%, t_50%, and t_75%, times to reach a 25%, 50%, and 75% cumulative release, respectively; MDT, mean dissolution time; DE_2hr, dissolution efficiency at 2 hours.

Figure 5 Cell viability of the HaCaT cells after being treated with various nanoparticle formulations and positive controls including clobetasol propionate as well as phenol solutions with and without 830-nm irradiation at 20 J/cm² (n = 4).

Abbreviations: PEm, Phyllanthus emblica L. fruit extract; IR, IR-820.

Figure 6 TEM images of (A) TRITC-Dex40-IR-NP, (B) TRITC-Dex40, (C) Intensity-weighted hydrodynamic size distribution, (D) Correlograms of TRITC-Dex40-IR-NP before and after SEC, and (E) Absorption spectra of NP, TRITC-Dex40-NP, and TRITC-Dex40-IR-NP. SEC f1 and SEC f2 are the first and second fractions eluted from SEC, respectively. The scale bars are displayed on each image.

Abbreviations: TEM, transmission electron microscope; TRITC-Dex40, tetramethylrhodamine isothiocyanate-dextran (molecular weight = 40kDa); IR, IR-820; SEC, size exclusion chromatography; f1, the first fraction; f2, the second fraction.

Figure 7 (A) Bright-field and fluorescent images of the HaCaT cells at 0, 2, and 6 hours post treatment with free TRITC-Dex40, TRITC-Dex40-NP, or TRITC-Dex40-IR-NP maintaining the concentration of TRITC-Dex40 at 9.4 μg/mL or IR-820 at 600 nM, and (B) Relative fold change in the fluorescence intensity of TRITC-Dex40 in the lysate extracted from the treated HaCaT cells. Different letters above the box plots indicate the statistically significant differences (P < 0.05) and n = 5. Scale bar = 100 µm.

Abbreviations: TRITC-Dex40, tetramethylrhodamine isothiocyanate-dextran (molecular weight = 40kDa); IR, IR-820.

Figure 8 (A) Timeline of the experiments for monitoring endolysosomal escape and deconvoluted confocal microscopy images of the HaCaT keratinocytes at 0, 2, and 4 hours post 6-hour treatment with (B, C) TRITC-Dex40-NP and (D, E) TRITC-Dex40-IR-NP (B, D) without and (C, E) with 830-nm irradiation at fluence of 20 J/cm². Scale bar = 10 µm.

Abbreviations: TRITC-Dex40, tetramethylrhodamine isothiocyanate-dextran (molecular weight = 40kDa); IR, IR-820; LAMP1, lysosomal-associated membrane protein 1; GFP, green fluorescent protein.

Figure 9 (A) Van Steensel CCFs, (B) ICQ, and (C) TOS_h ratios (irradiated to non-irradiated conditions) of the fluorescent images acquired from the CellLight^TM-lysosome-GFP-transfected HaCaT cells at 0, 2, and 4 hours post-treatment with TRITC-Dex40-IR-NP or TRITC-Dex40-NP for six hours before 830-nm irradiation at a fluence of 20 J/cm². n = 4 for (B, C).

Abbreviations: CCF, cross-correlation coefficient; ICQ, intensity correlation quotient; TOS_h, threshold overlap score at the highest threshold; GFP, green fluorescent protein; irr, irradiation; TRITC-Dex40, tetramethylrhodamine isothiocyanate-dextran (molecular weight = 40kDa); IR, IR-820.

Figure 10 Negative-stained TEM images of (A) IR-NP, (B) BCIP-IR-NP, (C) corresponding intensity-weighted hydrodynamic size distribution, (D) correlograms, and (E) absorption spectra. Scale bar = 100 nm.

Abbreviations: TEM, transmission electron microscope; IR, IR-820; BCIP, 5-bromo-4-chloro-3-indolyl phosphate disodium salt.

Figure 11 Ultra-thin section of the TEM images of the HaCaT keratinocytes treated with IR-NP and BCIP-IR-NP with and without 830-nm irradiation at 20 J/cm² post-treatment. Possible dehydroindigo precipitates are indicated by blue arrows. The scale bars are displayed on each image.

Abbreviations: N, nucleus; L, lysosome or endolysosome; V, vacuole; M, mitochondria; TEM, transmission electron microscope; IR, IR-820; BCIP, 5-bromo-4-chloro-3-indolyl phosphate disodium salt.