Innovative Microemulsion Loaded with Unusual Dimeric Flavonoids from Fridericia platyphylla (Cham.) L.G. Lohmann Roots.

Fridericia platyphylla (Cham.) L.G. Lohmann is a species native to the Brazilian cerrado, with promising bioactivity. The organic fraction of the roots is rich in unusual dimeric flavonoids, reported as potential candidates for cancer treatment. The exploration of these flavonoids is very important, considering their diverse biological activities and the need for innovative therapeutic options. This work aimed to develop and characterize a microemulsion loaded with a non-polar fraction (DCM). The constituents were chosen, and the pseudo-ternary diagram was constructed to determine the region of microemulsion formation. The microemulsions blank (ME), with 3% (ME3) and 5% (ME5) of fraction DCM, were characterized in terms of droplet size, zeta potential, and polydispersity index. Both MEs showed particle sizes <100 nm; only ME3 exhibited better values for polydispersity index and zeta potential and was therefore selected for further study. The organoleptic and physicochemical characteristics were evaluated, revealing limpidity and transparency typical of these microstructures, physiologically acceptable pH, refractive index of 1.42±0.01, and density of 1.017 g/cm3±0.01. The stability tests showed good stability profiles even after exposure to extreme thermal conditions, with minimal changes in pH and the content of the incorporated fraction. The in vitro release study demonstrated that ME3 enabled the controlled release of the fraction, with a cumulative amount released over 60% within 6 h. Furthermore, fraction DCM and ME3 exhibited no toxicity in Tenebrio molitor larvae. The developed microemulsion exhibited excellent properties, so this study represents the first successful attempt to develop a formulation that incorporates the dimeric flavonoid fraction.

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.

Design of Liquid Formulation Based on F127-Loaded Natural Dimeric Flavonoids as a New Perspective Treatment for Leishmaniasis.

Infectious and Parasitic Diseases (IPD) remain a challenge for medicine due to several interconnected reasons, such as antimicrobial resistance (AMR). American tegumentary leishmaniasis (ATL) is an overlooked IPD causing persistent skin ulcers that are challenging to heal, resulting in disfiguring scars. Moreover, it has the potential to extend from the skin to the mucous membranes of the nose, mouth, and throat in both humans and various animals. Given the limited effectiveness and AMR of current drugs, the exploration of new substances has emerged as a promising alternative for ATL treatment. Arrabidaea brachypoda (DC). Bureau is a native Brazilian plant rich in dimeric flavonoids, including Brachydin (BRA), which displays antimicrobial activity, but still little has been explored regarding the development of therapeutic formulations. In this work, we present the design of a low-cost liquid formulation based on the use of Pluronic F127 for encapsulation of high BRA concentration (LF-B500). The characterization techniques revealed that BRA-loaded F127 micelles are well-stabilized in an unusual worm-like form. The in vitro cytotoxicity assay demonstrated that LF-B500 was non-toxic to macrophages but efficient in the inactivation of forms of Leishmania amazonensis promastigotes with IC50 of 16.06 µg/mL. The results demonstrated that LF-B500 opened a new perspective on the use of liquid formulation-based natural products for ATL treatment.

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.

Design and Optimization of Stimuli-responsive Emulsion-filled Gel for Topical Delivery of Copaiba Oil-resin.

This study presents a phytotherapeutic emulsion-filled gel design composed of Pluronic® F127, Carbopol® C934P, and high level of copaiba oil-resin (PHY-ECO). Mathematical modeling and response surface methodology (RSM) were employed to access the optimal ratio between the oil and the polymer gel-matrix constituents. The chemometric approach showed robust mechanical and thermoresponsive properties for emulsion gel. The model predicts viscosity parameters at 35.0°C (skin temperature) from PHY-ECOs. Optimized PHY-ECOs were described by 18-20% (w/w) F127, 0.25% (w/w) C934P, and 15% (w/w) copaiba oil-resin, and showed interfacial layers properties that led to high physicochemical stability. Besides, it had thermal stimuli-responsive that led large viscosity range before and after skin administration, observed by oscillatory rheology. These behaviors give the optimized smart PHY-ECO high design potential to be used as a pharmaceutical platform for CO delivery, focusing on the anti-inflammatory therapy and skin wound care.

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.

Nanoencapsulated hypericin in P-123 associated with photodynamic therapy for the treatment of dermatophytosis.

The antifungal application of photodynamic therapy (PDT) has been widely explored. According to superficial nature of tinea capitis and the facility of application of light sources, the use of nanoencapsulated hypericin in P-123 associated with PDT (P123-Hy-PDT) has been a poweful tool to treat this pathology. Thus, the aim of this study was to evaluate the efficiency of P123-Hy-PDT against planktonic cells and in a murine model of dermatophytosis caused by Microsporum canis. In vitro antifungal susceptibility and in vivo efficiency tests were performed, including a skin toxicity assay, analysis of clinical signs by evaluating score, and photoacoustic spectroscopy. In addition, tissue analyses by histopathology and levels of pro-inflammatory cytokines, such as quantitative and qualitative antifungal assays, were employed. The in vitro assays demonstrated antifungal susceptibility with 6.25 and 12.5 µmol/L P123-Hy-PDI; these experiments are the first that have used this treatment of animals. P123-Hyp-mediated PDT showed neither skin nor biochemical alteration in vivo; it was safe for dermatophytosis treatment. Additionally, the treatment revealed rapid improvement in clinical signs at the site of infection after only three treatment sessions, with a clinical score confirmed by photoacoustic spectroscopy. The mycological reduction occurred after six treatment sessions, with a statistically significant decrease compared with untreated infected animals. These findings showed that P123-Hy-PDT restored tissue damage caused by infection, a phenomenon confirmed by histopathological analysis and proinflammatory cytokine levels. Our results reveal for the first time that P123-Hy-PDT is a promising treatment for tinea capitis and tinea corporis caused by M. canis, because it showed rapid clinical improvement and mycological reduction without causing toxicity.

Selective Photodynamic Effects on Breast Cancer Cells Provided by p123 Pluronic®- Based Nanoparticles Modulating Hypericin Delivery.

BACKGROUND: Breast cancer is the most relevant type of cancer and the second cause of cancer- related deaths among women in general. Currently, there is no effective treatment for breast cancer although advances in its initial diagnosis and treatment are available. Therefore, the value of novel anti-tumor therapeutic modalities remains an immediate unmet need in clinical practice. Following our previous work regarding the properties of the Pluronics with different photosensitizers (PS) for photodynamic therapy (PDT), in this study we aimed to evaluate the efficacy of supersaturated hypericin (HYP) encapsulated on Pluronic® P123 (HYP/P123) against breast cancer cells (MCF-7) and non-tumorigenic breast cells (MCF-10A). METHODS: Cell internalization and subcellular distribution of HYP/P123 was confirmed by fluorescence microscopy. The phototoxicity and citototoxicity of HYP/P123 was assessed by trypan blue exclusion assay in the presence and absence of light. Long-term cytotoxicity was performed by clonogenic assay. Cell migration was determined by the wound-healing assay. Apoptosis and necrosis assays were performed by annexin VFITC/ propidium Iodide (PI) by fluorescence microscopy. RESULTS: Our results showed that HYP/P123 micelles had high stability and high rates of binding to cells, which resulted in the selective internalization in MCF-7, indicating their potential to permeate the membrane of these cells. Moreover, HYP/P123 micelles accumulated in mitochondria and endoplasmic reticulum organelles, resulting in the photodynamic cell death by necrosis. Additionally, HYP/P123 micelles showed effective and selective time- and dose dependent phototoxic effects on MCF-7 cells but little damage to MCF-10A cells. HYP/P123 micelles inhibited the generation of cellular colonies, indicating a possible capability to prevent the recurrence of breast cancer. We also demonstrated that HYP/P123 micelles inhibit the migration of tumor cells, possibly by decreasing their ability to form metastases. CONCLUSION: Taken together, the results presented here indicate a potentially useful role of HYP/P123 micelles as a platform for HYP delivery to more specifically and effectively treat human breast cancers through photodynamic therapy, suggesting they are worthy for in vivo preclinical evaluations.

Theranostic verteporfin- loaded lipid-polymer liposome for photodynamic applications.

In this study we report a novel theranostic lipid-polymer liposome, obtained from DPPC and the triblock copolymer F127 covalently modified with 5(6)-carboxyfluorescein (CF) for photodynamic applications. Due to the presence of F127, small unilamellar vesicle (SUV) liposomes were synthesized by a simple and fast thin-film hydration method without the need for an extrusion process. The vesicles have around 100 nm, low polydispersity and superb solution stability. The clinically used photosensitizer verteporfin (VP) was entrapped into the vesicles, mostly in monomeric form, with 90% loading efficiency. Stern-Volmer and fluorescence lifetime assays showed heterogeneous distribution of the VP and CF into the vesicles, ensuring the integrity of their individual photophysical properties. The theranostic properties were entirely photoactivatable and can be trigged by a unique wavelength (470 nm). The feasibility of the system was tested against the Glioblastoma multiforme cell line T98G. Cellular uptake by time-resolved fluorescence microscopy showed monomerized VP (monoexponential decay, 6.0 ns) at nucleus level, while CF was detected at the membrane by fluorescence microscopy. The strategy's success was supported by the reduction of 98% in the viability of T98G cells by the photoactivated lipid-polymer liposome with [VP] = 1.0 µmol L-1. Therefore, the novel theranostic liposome is a potential system for use in cancer and ocular disease therapies.

Selective photodynamic effects on cervical cancer cells provided by P123 Pluronic®-based nanoparticles modulating hypericin delivery.

At present, cervical cancer is the fourth leading cause of cancer among women worldwide with no effective treatment options. In this study we aimed to evaluate the efficacy of hypericin (HYP) encapsulated on Pluronic® P123 (HYP/P123) photodynamic therapy (PDT) in a comprehensive panel of human cervical cancer-derived cell lines, including HeLa (HPV 18-positive), SiHa (HPV 16-positive), CaSki (HPV 16 and 18-positive), and C33A (HPV-negative), compared to a nontumorigenic human epithelial cell line (HaCaT). Were investigated: (i) cell cytotoxicity and phototoxicity, cellular uptake and subcellular distribution; (ii) cell death pathway and cellular oxidative stress; (iii) migration and invasion. Our results showed that HYP/P123 micelles had effective and selective time- and dose-dependent phototoxic effects on cervical cancer cells but not in HaCaT. Moreover, HYP/P123 micelles accumulated in endoplasmic reticulum, mitochondria and lysosomes, resulting in photodynamic cell death mainly by necrosis. HYP/P123 induced cellular oxidative stress mainly via type II mechanism of PDT and inhibited cancer cell migration and invasion mainly via MMP-2 inhibition. Taken together, our results indicate a potentially useful role of HYP/P123 micelles as a platform for HYP delivery to more specifically and effectively treat cervical cancers through PDT, suggesting they are worthy for in vivo preclinical evaluations.

Selective photodynamic effects on cervical adenocarcinoma cells provided by F127 Pluronic®-based micelles modulating hypericin delivery

Abstract Cervical cancer is a leading cause of death among women. The endocervical adenocarcinoma (ECA) represents an aggressive and metastatic type of cancer with no effective treatment options currently available. We evaluated the antitumoral and anti-migratory effects of hypericin (HYP) encapsulated on Pluronic F127 (F127/HYP) photodynamic therapy (PDT) against a human cell line derived from invasive cervical adenocarcinoma (HeLa) compared to a human epithelial cell line (HaCaT). The phototoxicity and cytotoxicity of F127/HYP were evaluated by the following assays: colorimetric assay, MTT, cellular morphological changes by microscopy and long-term cytotoxicity by clonogenic assay. In addition, we performed fluorescence microscopy to analyze cell uptake and subcellular distribution of F127/HYP, cell death pathway and reactive oxygen species (ROS) production. The PDT mechanism was determined with sodium azide and D-mannitol and cell migration by wound-healing assay. The treatment with F127/HYP promoted a phototoxic result in the HeLa cells in a dose-dependent and selective form. Internalization of F127/HYP was observed mainly in the mitochondria, causing cell death by necrosis and ROS production especially by the type II PDT mechanism. Furthermore, F127/HYP reduced the long-term proliferation and migration capacity of HeLa cells. Overall, our results indicate a potentially application of F127/HYP micelles as a novel approach for PDT with HYP delivery to more specifically treat ECA.

Response surface method optimization of a novel Hypericin formulation in P123 micelles for colorectal cancer and antimicrobial photodynamic therapy.

The photodynamic properties of Hypericin (Hyp) may be used as an alternative treatment for malignancies of the lower gastrointestinal tract and for the prevention of surgical-site infection; however, its use in photodynamic therapy has been limited because of its poor hydrosolubility. Therefore, in order to improve its water solubility and its photodynamic effect, Hyp was encapsulated in Pluronic P123 (P123) and the photodynamic effects against intestinal and epidermal bacteria and against two lineages of intestinal colon carcinoma cells were investigated. Two response surface methods (RSM) were used to achieve the best in vitro photodynamic activity against Enterococcus faecalis, Escherichia coli and Staphylococcus aureus: in the first (full 23 RSM), Hyp concentration (HC*), incubation time (IT*) and LED-light time (LT*) were considered as the independent variables and E. faecalis inhibition as the dependent variable. In the second (full 32 RSM), Hyp concentration (HC*) and P123 concentration (CC*) were considered as independent variables and E. faecalis, E. coli and S. aureus inhibition as dependent variables. The optimized experimental conditions achieved were: Hyp concentration=37.5µmol/L; P123 concentration=21.5 µmol/L and 6.3J/cm2, which resulted in 2.86±0.12 and 2.30±0.31CFU log-reductions of E. faecalis and S. aureus. No effect was seen against E. coli. The cytotoxic effects of Hyp/P123 were also investigated for Caco-2 and HT-29 intestinal colon carcinoma cells at Hyp/P123 concentrations of 1, 0.5, 0.25 and 0.1µmol/L for Caco-2 cells and 4, 3, 2 and 1µmol/L for HT-29 cells. The cytotoxic concentrations for 50% (CC50) and 90% (CC90) of Hyp/P123 were 0.443 and 0.870µmol/L for Caco-2 cells and 1.4 and 2.84µmol/L for HT-29 cells. The P123 nanocarrier played a significant role in the permeation of Hyp through the cell membrane leading to significant cell death, and showed itself to be a promising photosensitizer for PDT that could be suitable for the treatment of colonic diseases since it is effective against positive Gram bacteria and intestinal colon carcinoma cells.