Photodynamic therapy with various parietin formulations against breast cancer and bacteria
Parietin (PTN) has many benefits but presents great challenges in its clinical application since it possesses very lipophilic properties and forms aggregates in aqueous solutions. Various delivery systems, such as liposomes, polymeric nanoparticles, and cyclodextrin complexes, were tested regardi...
Saved in:
| Main Author: | |
|---|---|
| Contributors: | |
| Format: | Doctoral Thesis |
| Language: | English |
| Published: |
Philipps-Universität Marburg
2023
|
| Subjects: | |
| Online Access: | PDF Full Text |
| Tags: |
No Tags, Be the first to tag this record!
|
| Summary: | Parietin (PTN) has many benefits but presents great challenges in its clinical
application since it possesses very lipophilic properties and forms aggregates in
aqueous solutions. Various delivery systems, such as liposomes, polymeric
nanoparticles, and cyclodextrin complexes, were tested regarding the delivery of PTN
and photodynamic activity against bacteria and MDA-MB-231 cells.
The first and second projects focused on preparing PTN-loaded liposomes and
poly(lactic-co-glycolic acid) (PLGA) nanoparticles. The prepared particles were
subjected to physicochemical characterizations, such as entrapment efficiency, size,
zeta potential, release, and atomic force microscopy. The formulations were
monodispersed with sizes <200 nm and showed sufficient stability after storage in cell
culture medium and serum. Polymeric nanoparticles were superior in loading efficiency
and long-term stability compared to liposomes, particularly after lyophilization.
It is crucial to ensure the photoactivity of the formulations by investigating PTN's
potential to undergo type I (superoxide anion) and type II mechanisms (singlet oxygen)
in photodynamic therapy (PDT). Intriguingly, PTN exhibited higher singlet oxygen
quantum yield in liposomes than in polymeric nanoparticles. The proximity of
photosensitizer (PS) molecules because of high loading concentrations might result in
aggregation-caused self-quenching. Moreover, the outward diffusion of singlet oxygen
from the nanoparticles may be hampered by the polymeric matrix, but it could be easier
in the lipid bilayer of liposomes. Additionally, the formulations increased PTN's
photostability against blue light irradiation.
Furthermore, the formulations were investigated on MDA-MB-231 cells. Concentration
range, incubation time, and radiant exposure were studied, and in vitro phototoxicity
was assessed by MTT. Incubation for 4 h resulted in higher photokilling, which
coincided with flow cytometry result, where the highest uptake was seen after 4 h.
Based on these results, the PTN concentration range (0.1-10 μM), fluence level (6
J/cm2) and incubation time (4 h) were used in the study. The liposomes were shown to
have more cell killing (lower IC50) than PLGA nanoparticles due to higher singlet
oxygen quantum yield and probable higher uptake. The phototoxicity was also
confirmed by live/dead staining, where the cells were incubated with IC50 and stained
with calcein/propidium iodide. Confocal laser scanning microscopy showed that PTN was distributed into the
cytoplasm, specifically into the mitochondria and lysosome, after staining with
mitotracker and lysotracker. Afterward, the intracellular ROS generation was
qualitatively and quantitatively assessed, confirming a PDT dependency (presence of
both light and PS). Then, staining with specific dyes confirmed ROS-induced lysosomal
and mitochondrial membrane permeabilization in MDA-MB-231 cells. These results
were corroborated by flow cytometry after staining the treated cells with annexin VFITC/
propidium iodide, differentiating the apoptotic and necrotic cells from live ones.
The percentage of apoptotic and necrotic cells increased upon blue laser irradiation.
The biocompatibility of the formulation was investigated using hen's egg test
chorioallantoic membrane (HET-CAM), which showed safety with no irritation,
hemorrhage, lysis, or coagulation potential. The anti-angiogenic effect was studied on
the chorioallantoic membrane (CAM) model, and the results indicated that PDT
induced vascular response, apparent mild closure, and collapse of smaller capillaries
confined to the irradiated areas. Finally, the formulation was further analyzed in a
xenografted MDA-MB-231 tumor model. Positron emission tomography/computed
tomography (PET/CT) was used as an imaging technique to evaluate the tumor's
response after PDT by monitoring the glucose metabolism using fluorodeoxyglucose
(18F-FDG) as a radiotracer. The metabolic activity was remarkably decreased
compared to the negative control.
In the third project, cyclodextrin complexation (PTN/HP-β-CD) was investigated to
enhance the aqueous solubility of PTN. Various solid (1H NMR, FT-IR, x-ray diffraction,
scanning electron microscopy, and differential scanning calorimetry) and solutionbased
techniques (UV spectroscopy and singlet oxygen quantum yield) were used to
confirm the complexation of PTN in (2-hydroxypropyl)-β-cyclodextrin, which
successfully solubilized PTN (28-fold in water), retained its photodynamic activity in
aqueous vehicle and minimized its photodegradation.
Photodynamic inactivation using PTN/HP-β-CD was performed on gram-positive
(Staphylococcus saprophyticus subsp. bovis) and gram-negative (Escherichia coli
DH5α) bacteria. The bacterial viability of gram-positive bacteria was significantly
decreased by >4.2 log, while only a slight reduction in bacterial viability > 1.0 log was
observed in E. coli. |
|---|---|
| DOI: | 10.17192/z2023.0680 |