Hypericin photodynamic activity in DPPC liposomes - part II: stability and application in melanoma B16-F10 cancer cells.

Hypericin (Hyp) is considered a promising photosensitizer for Photodynamic Therapy (PDT), due to its high hydrophobicity, affinity for cell membranes, low toxicity and high photooxidation activity. In this study, Hyp photophysical properties and photodynamic activity against melanoma B16-F10 cells were optimized using DPPC liposomes (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) as a drug delivery system. This nanoparticle is used as a cell membrane biomimetic model and solubilizes hydrophobic drugs. Hyp oxygen singlet lifetime (τ) in DPPC was approximately two-fold larger than that in P-123 micelles (Pluronic™ surfactants), reflecting a more hydrophobic environment provided by the DPPC liposome. On the other hand, singlet oxygen quantum yield values (ΦΔ1O2) in DPPC and P-123 were similar; Hyp molecules were preserved as monomers. The Hyp/DPPC liposome aqueous dispersion was stable during fluorescence emission and the liposome diameter remained stable for at least five days at 30 °C. However, the liposomes collapsed after the lyophilization/rehydration process, which was resolved by adding the lyoprotectant Trehalose to the liposome dispersion before lyophilization. Cell viability of the Hyp/DPPC formulation was assessed against healthy HaCat cells and high-metastatic melanoma B16-F10 cells. Hyp incorporated into the DPPC carrier presented a higher selectivity index than the Hyp sample previously solubilized in ethanol under the illumination effect. Moreover, the IC50 was lower for Hyp in DPPC than for Hyp pre-solubilized in ethanol. These results indicate the potential of the formulation of Hyp/DPPC for future biomedical applications in PDT treatment.

Hypericin photodynamic activity. Part III: in vitro evaluation in different nanocarriers against trypomastigotes of Trypanosoma cruzi.

Chagas is a parasitic endemic disease caused by the protozoan Trypanosoma cruzi. It represents a strong threat to public health due to its strong resistance against commonly available drugs. We studied the in vitro ability to inactivate the trypomastigote form of this parasite using photodynamic inactivation of microorganisms (or antimicrobial Photodynamic Therapy, aPDT). For this, we chose to use the photosensitizer hypericin (Hyp) formulated in ethanol/water (1% v/v) and Hyp loaded in the dispersion of different aqueous nanocarrier systems. These included polymeric micelles of F-127 and P-123 (both Pluronic™ surfactants), and liposomal vesicles of phospholipid 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). These systems with Hyp had their activity compared against trypomastigote forms under light and in the dark. Hyp revealed a high level of effectiveness to eradicate protozoa in vitro. Samples at concentrations higher than 0.8 µmol L-1 of Hyp in Pluronic micelles showed efficacy even in the dark, with the EC50 around (6-8) µmol L-1. Therefore, Hyp/Pluronics can be used also as a chemotherapeutic agent. The best result for EC50 is at approximately 0.31 µmol L-1 for illuminated systems of Hyp in F-127 micelles. For Hyp in P-123 micelles under light, the results also led to a low EC50 value of 0.36 µmol L-1. The highest value of EC50 was 2.22 µmol L-1, which was found for Hyp/DPPC liposomes under light. For the Hyp-free (ethanol/water, 1% v/v)/illuminated group, the EC50 value was 0.37 µmol L-1, which also is a value that shows effectiveness. However, in free-form, Hyp is not protected against blood components, unlike when Hyp is loaded into the nanocarriers.

Advanced theranostic nanoplatforms for hypericin delivery in the cancer treatment.

Biomodified coated-lipid vesicles were obtained using the DPPC lipid (L) and F127 copolymer linked covalently with spermine (SN), biotin (BT), and folic acid (FA), resulting in LF127-SN, LF127-BT, and LF127-FA nanoplatforms. The photosensitizer hypericin (HY) was incorporated into the nanosystem by a thin-film method and characterized by dynamic light scattering, zeta potential, encapsulation efficiency, and transmission electronic microscopy. The results provided a good level of stability for all nanoplatforms for at least 5 days as an aqueous dispersion. The in vitro serum stability showed that the HY-loaded LF127-SN has a lower tendency to form complexes with BSA protein than with its analogs. LF127-SN was the most stable HY formulation, followed by LF127-BT and LF127-FA, confirmed by the association constant (Kd) values: 600 µmol L-1, 1100 µmol L-1, 515 µmol L-1, and 378 µmol L-1 for LF127, LF127 FA, LF127-BT, and LF127-SN, respectively. The photodynamic potential of HY was accessed by cytotoxicity assays using Caco-2, B16-F10, L-929, and HaCat cells. HY-loaded LF127-SN revealed a significant increase in the selectivity compared to other nanoplatforms. HY-loaded in LF127-BT and LF127-SN showed distinct uptake and biodistribution after 2 h of intravenous application. All biomodified coated-lipids showed satisfactory metabolism within 72 h after application, without significant accumulation or residue in any vital organ. These results suggest that incorporating HY-loaded in these nanosystems may be a promising strategy for future applications, even with a small amount of binders to the coating copolymer (0.02% w/v). Furthermore, these results indicate that the LF127-SN showed remarkable superiority compared to other evaluated systems, being the most distinct for future photodynamic therapy and theranostic applications.

Interaction of triblock copolymers (Pluronic®) with DMPC vesicles: a photophysical and computational study.

Pluronic/lipid mix promises stealth liposomes with long circulation time and long-term stability for pharmaceutical applications. However, the influence of Pluronics on several aspects of lipid membranes has not been fully elucidated. Herein it was described the effect of Pluronics on the structured water, alkyl chain conformation, and kinetic stability of dimyristoylphosphatidylcholine (DMPC) liposomes using interfacial and deeper fluorescent probes along with computational molecular modeling data. Interfacial water changed as a function of Pluronics' hydrophobicity with polypropylene oxide (PPO) anchoring the copolymers in the lipid bilayer. Pluronics with more than 30-40 PO units had facilitated penetration at the bilayer while shorter PPO favored a more interfacial interaction. Low Pluronic concentrations provided long-term stability of vesicles by steric effects of polyethylene oxide (PEO), but high amounts destabilized the vesicles as a sum of water-bridge cleavage at the polar head group and the reduced alkyl-alkyl interactions among the lipids. The high kinetic stability of Pluronic/DMPC vesicles is a proof-of-concept of its advantages and applicability in nanotechnology over conventional liposome-based pharmaceutical products for future biomedical applications.

Melanoma-targeted photodynamic therapy based on hypericin-loaded multifunctional P123-spermine/folate micelles.

Multifunctional P123 micelle linked covalently with spermine (SM) and folic acid (FA) was developed as a drug delivery system of hypericin (HYP). The chemical structures of the modified copolymers were confirmed by spectroscopy and spectrophotometric techniques (UV-vis, FTIR, and 1H NMR). The copolymeric micelles loading HYP were prepared by solid dispersion and characterized by UV-vis, fluorescence, dynamic light scattering (DLS), ζ potential, and transmission electron microscopy (TEM). The results provided a good level of stability for HYP-loaded P123-SM, P123-FA, and P123-SM/P123-FA in the aqueous medium. The morphology analysis showed that all copolymeric micelles are spherical. Well-defined regions of different contrast allow us to infer that SM and FA were localized on the surface of micelles, and the HYP molecules are located in the core region of micelles. The uptake potential of multifunctional P123 micelle was accessed by exposing the micellar systems loading HYP to two cell lines, B16-F10 and HaCaT. HYP-loaded P123 micelles reveal a low selectivity for melanoma cells, showing significant photodamage for HaCat cells. However, the exposition of B16-F10 cells to Hyp-loaded SM- and FA-functionalized P123 micelles under light irradiation revealed the lowest CC50 values. The interpretation of these results suggested that the combination of SM and FA on P123 micelles is the main factor in enhancing the HYP uptake by melanoma cells, consequently leading to its photoinactivation.

Characterization of chlorophyll derivatives in micelles of polymeric surfactants aiming photodynamic applications.

The spectrophotometric properties of chlorophylls' derivatives (Chls) formulated in the Pluronics® F-127 and P-123 were evaluated and the results have shown that the Chls were efficiently solubilized in these drug delivery systems as monomers. The relative location of the Chls in the Pluronics® was estimated from the Stokes shift and micropolarity of the micellar environment. Chls with phytyl chain were located in the micellar core, where the micropolarity is similar to ethanol, while phorbides' derivatives (without phytyl chain) were located in the outer shell of the micelle, i.e., more polar environment. In addition, the thermal stability of the micellar formulations was evaluated through electronic absorption, fluorescence emission and resonance light scattering with lowering the temperature. The Chls promote the stability of the micelles at temperatures below the Critical Micellar Temperature (CMT) of these surfactants. For F-127 formulations, the water molecules drive through inside the nano-structure at temperatures below the CMT, which increased the polarity of this microenvironment and directly affected the spectrophotometric properties of the Chls with phytyl chain. The properties of the micellar microenvironment of P-123, with more hydrophobic core due to the small PEO/PPO fraction, were less affected by lowering the temperature than for F-127. These results enable us to better understand the Chls behavior in micellar copolymers and allowed us to design new drug delivery system that maintains the photosensitizer's properties for photodynamic applications.