Stabilized tetraether lipids based particles guided prophyrins photodynamic therapy

Figures & data

Figure 1. Fluorescence spectra of PpIX in various TEL-liposomes and their freeze-fractured micrographs (A). The fluorescence of PpIX in TEL_9mol% (a₁), TEL_29.9 mol% (a₂) and TEL_62mol% (a₃) based liposomes for TL: PpIX 10 (

and 100

Freeze-fracture micrographs of PpIX manifested well-segregated vesicular structures in TEL_9mol%, (a_1-2), TEL_29.9 mol% (a_2-2) and TEL_62mol%(a_3-2) based liposomes. Part B illustrates the in-vitro time-dependent fluorescence recovery of PpIX from TEL_PpIX-Donor based TEL_9mol%, (black solid circles), TEL_29.2 mol% (blue solid circles) or TEL_62mol% (green solid circles) liposomes after incubation with DPPC_{PpIX-Acceptor} liposomes (b₁) or HSA_{PpIX-Acceptor} (b₂). Values at the curves stand for the calculated TL: PpIX ratios during PpIX transfer. Data were shown as mean ± standard deviation of three independent experiments.

Figure 2. Photo cytotoxicity and dose-dependent effect of PpIX in SKOV-3 (A) and L929 (B) cells. After incubation with TEL_9mol% liposomes (a₁ and b₁) or TEL_62mol% (a₂ and b₂) liposomes at TL: PpIX 10 (t_PpIX = 3h), cells were light irradiated at a series of radiant exposure doses of 134, 202, 403 and 672mJ.cm⁻² (irradiance = 22 W.m⁻²). Cell survival is represented as a percentage of control-cell growth in cultures containing no PpIX. Cell proliferation was quantified colorimetrically using MTT assay. Half-maximal inhibitory concentrations (IC₅₀) for PpIX were calculated from the fitted dose–response curves. In graph a₃, elevated generation of singlet oxygen in SKOV-3 after various cPDT treatments with PpIX at the same radiant exposure doses is presented. Each point represents the mean of quintuplicates (n = 5) ±SD of three separate experiments; ***p < .001.

Figure 3. Confocal laser scanning acquisitions of L929 cells treated with various TEL-liposomes at PpIX =25 µM. Image acquisitions of TEL_{9mol %} liposomes (a_1(t=1h), a_2(t=3h)) and TEL_{62mol %} liposomes (b_1(t=1h), b_2(t=3h)) and free PpIX (c_1(t=1h), c_2(t=3h)) are presented. (1) DAPI localized nuclei images, (2) empty channel, (3) PpIX fluorescence images and (4) merged images (magnified, right panel). Z-Stack mode acquisition of b₂ shows the cellular co-localization of PpIX in the cytoplasm after incubation with TEL_62mol% liposomes (t_PpIX=3h). The total stack size was 9.5 μm with scale of 0.5 μm each.

Table 1. Particle size and zeta potential measurements of TEL-liposomes.

TEL mol% in liposomes (TL: PpIX ratios)		Particle size ± SD nm (PDI)		Zeta-potential ± SD mV
TEL0mol%
PpIX-free liposomes		172.3 ± 1.3 (0.1)		+3.0 ± 0.1
(100)		134.2 ± 0.6 (0.1)		−22.9 ± 3.0
(10)		186.3 ± 3.6 (0.3)		−42.0 ± 3.3
TEL9mol%
PpIX-free liposomes		169.4 ± 2.9 (0.2)		−20.5 ± 0.6
(100)		139.3 ± 1.4 (0.1)		−34.1 ± 1.5
(10)		173.0 ± 2.6 (0.3)		−38.3 ± 4.1
TEL29.9mol%
PpIX-free liposomes		207.1 ± 18.0 (0.3)		−34.7 ± 3.52
(100)		142.6 ± 0.4 (0.1)		−36.1 ± 1.5
(10)		256 ± 6.1 (0.3)		−41.9 ± 2.17
TEL62mol%
PpIX-free liposomes		194.0 ± 3.0 (0.4)		−40.3 ± 0.5
(100)		163.5 ± 2.0 (0.2)		−39.8 ± 3.9
(10)		176.5 ± 2.4 (0.2)		−44.1 ± 0.2

Table 2. In vitro PpIX release kinetics and mechanisms from TEL-liposomes.

		Kinetic of PpIX Release						Mechanism of PpIX Release
Kinetic Modesl (Dash et al., 2010)		Zero Order			First Oder			Diffusion (Higuchi Model)			Baker & Lonsdale			Korsmeyer–Peppas
In-Vitro PpIX release kinetics and mechanisms from TEL-liposomes (donor) to PpIX-free DPPC liposomes (acceptor)
Kinetic Equation		${\mathit{M}}_{\mathit{t}}/{\mathit{M}}_{\mathit{\infty }}={\mathit{K}}_0\mathit{t}$			$\mathit{ln}\mathit{ }(1-{\mathit{M}}_{\mathit{t}}/{\mathit{M}}_{\mathit{\infty }})=-{\mathit{K}}_1\mathit{t}\mathit{ }$			${\mathit{M}}_{\mathit{t}}/{\mathit{M}}_{\mathit{\infty }}={\mathit{K}}_{\mathit{H}}{\mathit{t}}^{1/2}$			$3/2\mathit{ }\left[1-\left(1-\right.\right.\left.{\mathit{M}}_{\mathit{t}}/{\mathit{M}}_{\mathit{\infty }}\right)\left.2/3\right]$ $-{\mathit{M}}_{\mathit{t}}/{\mathit{M}}_{\mathit{\infty }}=\mathit{ }{\mathit{K}}_{\mathit{B}\mathit{L}}\mathit{t}$			${\mathit{M}}_{\mathit{t}}/{\mathit{M}}_{\mathit{\infty }}={\mathit{K}}_{\mathit{K}\mathit{P}}{\mathit{t}}^{\mathit{n}}$
Kinetic Parameters		r^2	K0		r^2	K1		r^2	KH		r^2	KB&L		r^2	n	Kkp
TEL9 mol%		0.88393	15.82463		0.89597	0.22856		0.98241	16.56128		0.91763	0.02055		0.99040	0.11200	38.17684
TEL29.9 mol%		0.99110	0.81309		0.99347	0.01158		0.99102	4.78710		0.99221	0.00101		0.94090	0.21090	18.90166
TEL62 mol%		0.98729	1.18327		0.99199	0.01577		0.99714	7.03667		0.99624	0.00118		0.98330	0.40390	10.26124
In-Vitro PpIX release kinetics and mechanisms from TEL-liposomes (donor) to HSA (acceptor)
TEL9 mol%		0.95042	0.25853		0.95249	0.00342		0.98518	1.57791		0.95789	0.00025		0.99280	0.08520	20.80176
TEL29.9 mol%		0.92609	0.34173		0.93003	0.00405		0.98036	1.91829		0.94822	0.00020		0.99420	0.13450	12.67651
TEL62 mol%		0.95862	0.35997		0.96077	0.00394		0.99045	1.85219		0.97866	0.00011		0.93020	0.21780	5.96624

M_t/M₀: The fraction of drug released at time t; K₀: Zero order release rate constant; t: The release time; KO₁: First order release rate constant; r²: Correlation Coefficient Squared; K_H: Higuchi release rate constant; n: The parameter that depends on the release mechanism; K_B&L: Baker & Lonsdale release rate constant; KKP: Korsmeyer–Peppas release rate constant.

Figure 4. Stereomicrographs of CAM represent the occlusion of CAM vasculature mediated photodynamic therapy (vPDT). Image acquisitions were performed at 5 minutes before PDT (t_post 0) and at t_post 5, 30, and 60 minutes after PDT following IV-injection of free PpIX in 20% PEG 16000 containing HEPES-buffered saline, PpIX in TEL_9mol% or in TEL_62mol% liposomes at TL: PpIX ratios of 10. Bar represents 500 μm.