Essential oil of oregano (Origanum vulgare L.) reduces infection and proliferation of Toxoplasma gondii in BeWo cells with induction of autophagy and death of tachyzoites through a mechanism similar to necrosis.

Toxoplasmosis poses a global health threat, ranging from asymptomatic cases to severe, potentially fatal manifestations, especially in immunocompromised individuals and congenital transmission. Prior research suggests that oregano essential oil (OEO) exhibits diverse biological effects, including antiparasitic activity against Toxoplasma gondii. Given concerns about current treatments, exploring new compounds is important. This study was to assess the toxicity of OEO on BeWo cells and T. gondii tachyzoites, as well as to evaluate its effectiveness in in vitro infection models and determine its direct action on free tachyzoites. OEO toxicity on BeWo cells and T. gondii tachyzoites was assessed by MTT and trypan blue methods, determining cytotoxic concentration (CC50), inhibitory concentration (IC50), and selectivity index (SI). Infection and proliferation indices were analyzed. Direct assessments of the parasite included reactive oxygen species (ROS) levels, mitochondrial membrane potential, necrosis, and apoptosis, as well as electron microscopy. Oregano oil exhibited low cytotoxicity on BeWo cells (CC50: 114.8 µg/mL ± 0.01) and reduced parasite viability (IC50 12.5 ± 0.06 µg/mL), demonstrating 9.18 times greater selectivity for parasites than BeWo cells. OEO treatment significantly decreased intracellular proliferation in infected cells by 84% after 24 h with 50 µg/mL. Mechanistic investigations revealed increased ROS levels, mitochondrial depolarization, and lipid droplet formation, linked to autophagy induction and plasma membrane permeabilization. These alterations, observed through electron microscopy, suggested a necrotic process confirmed by propidium iodide labeling. OEO treatment demonstrated anti-T. gondii action through cellular and metabolic change while maintaining low toxicity to trophoblastic cells.

Synthetic dibenzylideneketones as promising anti-herpes simplex virus type 1 agents.

New antiviral agents for the treatment of herpes simplex virus type 1 (HSV-1) infection, which causes a highly prevalent and incurable disease, are needed. Here, we report for the first time the in vitro anti-HSV-1 activity of two dibenzylideneketone compounds: DBK1 and DBK2. DBK1 demonstrated virucidal activity, and high-resolution scanning electron microscopy showed that it caused morphological changes in the HSV-1 envelope. DBK2 was able to reduce HSV-1 plaque size in vitro. The DBKs are promising anti-HSV-1 candidates, as they exhibit low toxicity and exert an antiviral effect by acting at the early stages of HSV-1-host cell interaction.

Natural compounds based chemotherapeutic against Chagas disease and leishmaniasis: mitochondrion as a strategic target.

Over the past years, natural products have been explored in order to find biological active substances to treat various diseases. Regarding their potential action against parasites such as trypanosomatids, specially Trypanosoma cruzi and Leishmania spp., much advance has been achieved. Extracts and purified molecules of several species from genera Piper, Tanacetum, Porophyllum, and Copaifera have been widely investigated by our research group and exhibited interesting antitrypanosomal and antileishmanial activities. These natural compounds affected different structures in parasites, and we believe that the mitochondrion is a strategic target to induce parasite death. Considering that these trypanosomatids have a unique mitochondrion, this cellular target has been extensively studied aiming to find more selective drugs, since the current treatment of these neglected tropical diseases has some challenges such as high toxicity and prolonged treatment time. Here, we summarise some results obtained with natural products from our research group and we further highlighted some strategies that must be considered to finally develop an effective chemotherapeutic agent against these parasites.

Moringa oleifera extract promotes apoptosis-like death in Toxoplasma gondii tachyzoites in vitro.

Toxoplasma gondii is the causative agent of toxoplasmosis, and an important problem of public health. The current treatment for toxoplasmosis is the combination of pyrimethamine and sulphadiazine, which do not act in the chronic phase of toxoplasmosis and have several side-effects. This study evaluated the anti-T. gondii activity and potential mechanism of Moringa oleifera seeds' aqueous extract in vitro. The concentration of M. oleifera extract in HeLa cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability assays. The presence of T. gondii was assessed by quantitative polymerase chain reaction and toluidine blue staining. Pyrimethamine and sulphadiazine were used as drug controls. Modifications in T. gondii morphology and ultrastructure were observed by electron microscopy. In vitro, the M. oleifera extract had no toxic effect on HeLa cells at concentrations below 50 µg mL−1. Moringa oleifera extract inhibits T. gondii invasion and intracellular proliferation with similar results for sulphadiazine + pyrimethamine, and also shows cellular nitric oxide production at a concentration of 30 µg mL−1. Electron microscopy analyses indicated structural and ultrastructural modifications in tachyzoites after treatment. We also observed an increase in reactive oxygen species production and a loss of mitochondrial membrane integrity. Nile Red staining assays demonstrated a lipid accumulation. Annexin V­fluorescein isothiocyanate and propidium iodide staining demonstrated that the main action of M. oleifera extract in T. gondii tachyzoites was compatible with late apoptosis. In conclusion, M. oleifera extract has anti-T. gondii activity in vitro and might be a promising substance for the development of a new anti-T. gondii drug.

Discovery of 1,3,4,5-tetrasubstituted pyrazoles as anti-trypanosomatid agents: Identification of alterations in flagellar structure of L. amazonensis.

Trypanosoma cruzi and Leishmania species are causative agents of Chagas disease and Leishmaniasis, respectively, known as Neglected Tropical Diseases. Up to now, the treatments are inadequate and based on old drugs. Thus, we report herein the discovery of 1,3,4,5-tetrasubstituted pyrazole derivatives that presented potent and selective inhibition against promastigote forms of L. amazonensis, and epimastigote forms of T. cruzi. The structure-activity relationship led to the identification of three compounds (2m, 2n and 2p) with an in vitro IC50 of 7.4 µM (selective index - SI ≥ 133.0), 3.8 µM (SI in the range of 148.4 to 200.8), and 7.3 µM (SI in the range of 87.2 to 122.4) against L. amazonensis, respectively. Also, those compounds exhibited in vitro IC50 of 9.7 µM (SI ≥ 101.5), 4.5 µM (SI in the range of 125.3 to 169.6) and 17.1 µM (SI in the range of 37.2 to 52.2) against T. cruzi, respectively. A preliminary study about the reaction mechanism in promastigotes showed that 2n caused an increase of the production of ROS and of lipid storage bodies. Furthermore, 2n induced abnormalities in the flagellum that may have an impact on the parasite motility.

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.

Dibenzylideneacetones Are Potent Trypanocidal Compounds That Affect the Trypanosoma cruzi Redox System.

Despite ongoing efforts, the available treatments for Chagas' disease are still unsatisfactory, especially in the chronic phase of the disease. Our previous study reported the strong trypanocidal activity of the dibenzylideneacetones A3K2A1 and A3K2A3 against Trypanosoma cruzi (Z. Ud Din, T. P. Fill, F. F. de Assis, D. Lazarin-Bidóia, V. Kaplum, F. P. Garcia, C. V. Nakamura, K. T. de Oliveira, and E. Rodrigues-Filho, Bioorg Med Chem 22:1121-1127, 2014, http://dx.doi.org/10.1016/j.bmc.2013.12.020). In the present study, we investigated the mechanisms of action of these compounds that are involved in parasite death. We showed that A3K2A1 and A3K2A3 induced oxidative stress in the three parasitic forms, especially trypomastigotes, reflected by an increase in oxidant species production and depletion of the endogenous antioxidant system. This oxidative imbalance culminated in damage in essential cell structures of T. cruzi, reflected by lipid peroxidation and DNA fragmentation. Consequently, A3K2A1 and A3K2A3 induced vital alterations in T. cruzi, leading to parasite death through the three pathways, apoptosis, autophagy, and necrosis.

Unsymmetrical 1,5-diaryl-3-oxo-1,4-pentadienyls and their evaluation as antiparasitic agents.

In this work the synthesis and antiparasitical activity of new 1,5-diaryl-3-oxo-1,4-pentadienyl derivatives are described. First, compounds 1a, 1b, 1c and 1d were prepared by acid-catalyzed aldol reaction between 2-butanone and benzaldehyde, anisaldehyde, p-N,N-dimethylaminobenzaldehyde and p-nitrobenzaldehyde. Reacting each of the methyl ketones 1a, 1b, 1c and 1d with the p-substituted benzaldehydes under basic-catalyzed aldol reaction, we further prepared compounds 2a-2p. All twenty compounds were evaluated for antiproliferative activity, particularly for promastigote of Leishmania amazonensis and epimastigote of Trypanosoma cruzi. All compounds showed good activity while nitro compounds 2i and 2k showed inhibition activity at a few µM.

Additional evidence of the trypanocidal action of (-)-elatol on amastigote forms through the involvement of reactive oxygen species.

Chagas' disease, a vector-transmitted infectious disease, is caused by the protozoa parasite Trypanosoma cruzi. Drugs that are currently available for the treatment of this disease are unsatisfactory, making the search for new chemotherapeutic agents a priority. We recently described the trypanocidal action of (-)-elatol, extracted from the macroalga Laurencia dendroidea. However, nothing has been described about the mechanism of action of this compound on amastigotes that are involved in the chronic phase of Chagas' disease. The goal of the present study was to evaluate the effect of (-)-elatol on the formation of superoxide anions (O2•-), DNA fragmentation, and autophagy in amastigotes of T. cruzi to elucidate the possible mechanism of the trypanocidal action of (-)-elatol. Treatment of the amastigotes with (-)-elatol increased the formation of O2•- at all concentrations of (-)-elatol assayed compared with untreated parasites. Increased fluorescence was observed in parasites treated with (-)-elatol, indicating DNA fragmentation and the formation of autophagic compartments. The results suggest that the trypanocidal action of (-)-elatol might involve the induction of the autophagic and apoptotic death pathways triggered by an imbalance of the parasite's redox metabolism.

Discovery of a new pyrido[2,3-d]pyridazine-2,8-dione derivative as a potential anti-inflammatory agent through COX-1/COX-2 dual inhibition.

In this paper, we present the design and synthesis of a novel series of pyrido[2,3-d]pyridazine-2,8-dione derivatives via the annulation of the 2-pyridone pattern. The synthesized derivatives were evaluated for in vivo anti-inflammatory activity using an ear edema model. Compound 7c, which showed a greater inhibition of ear edema (82%), was further tested for its in vitro COX-1/COX-2 inhibitory activity. Compound 7c showed similar inhibitory activities against COX-1 and COX-2 isoenzymes. The structural features that ensure the dual inhibition of COX-1 and COX-2 were elucidated using molecular docking studies. Overall, the ring closing of 2-pyridone pattern I transformed this highly selective COX-2 inhibitor into a dual COX inhibitor (7c), which could serve as a model for determining selectivity for COX-2.

Synthesis and Anti-Chagas Activity Profile of a Redox-Active Lead 3-Benzylmenadione Revealed by High-Content Imaging.

Chagas disease, or American trypanosomiasis, is a neglected tropical disease which is a top priority target of the World Health Organization. The disease, endemic mainly in Latin America, is caused by the protozoan Trypanosoma cruzi and has spread around the globe due to human migration. There are multiple transmission routes, including vectorial, congenital, oral, and iatrogenic. Less than 1% of patients have access to treatment, relying on two old redox-active drugs that show poor pharmacokinetics and severe adverse effects. Hence, the priorities for the next steps of R&D include (i) the discovery of novel drugs/chemical classes, (ii) filling the pipeline with drug candidates that have new mechanisms of action, and (iii) the pressing need for more research and access to new chemical entities. In the present work, we first identified a hit (4a) with a potent anti-T. cruzi activity from a library of 3-benzylmenadiones. We then designed a synthetic strategy to build a library of 49 3-(4-monoamino)benzylmenadione derivatives via reductive amination to obtain diazacyclic benz(o)ylmenadiones. Among them, we identified by high content imaging an anti-amastigote "early lead" 11b (henceforth called cruzidione) revealing optimized pharmacokinetic properties and enhanced specificity. Studies in a yeast model revealed that a cruzidione metabolite, the 3-benzoylmenadione (cruzidione oxide), enters redox cycling with the NADH-dehydrogenase, generating reactive oxygen species, as hypothesized for the early hit (4a).

3,3',5,5'-Tetramethoxybiphenyl-4,4'diol triggers oxidative stress, metabolic changes, and apoptosis-like process by reducing the PI3K/AKT/NF-κB pathway in the NCI-H460 lung cancer cell line.

Lung cancer is one of the leading causes of cancer-related deaths in men and women worldwide. Current treatments have limited efficacy, cause significant side effects, and cells can develop drug resistance. New therapeutic strategies are needed to discover alternative anticancer agents with high efficacy and low-toxicity. TMBP, a biphenyl obtained by laccase-biotransformation of 2,6-dimethoxyphenol, possesses antitumor activity against A549 adenocarcinoma cells. Without causing damage to sheep erythrocytes and mouse peritoneal macrophages of BALB/c mice. In addition to being classified as a good oral drug according to in-silico studies. This study evaluated the in-vitro cytotoxic effect of TMBP on lung-cancer cell-line NCI-H460 and reports mechanisms on immunomodulation and cell death. TMBP treatment (12.5-200 µM) inhibited cell proliferation at 24, 48, and 72 h. After 24-h treatment, TMBP at IC50 (154 µM) induced various morphological and ultrastructural changes in NCI-H460, reduced migration and immunofluorescence staining of N-cadherin and ß-catenin, induced increased reactive oxygen species and nitric oxide with reduced superoxide radical-anion, increased superoxide dismutase activity and reduced glutathione reductase. Treatment also caused metabolic stress, reduced glucose-uptake, intracellular lactate dehydrogenase and lactate levels, mitochondrial depolarization, increased lipid droplets, and autophagic vacuoles. TMBP induced cell-cycle arrest in the G2/M phase, death by apoptosis, increased caspase-3/7, and reduced STAT-3 immunofluorescence staining. The anticancer effect was accompanied by decreasing PI3K, AKT, ARG-1, and NF-κB levels, and increasing iNOS. These results suggest its potential as a candidate for use in future lung anticancer drug design studies.

Trilobolide-6-O-isobutyrate from Sphagneticola trilobata acts by inducing oxidative stress, metabolic changes and apoptosis-like processes by caspase 3/7 activation of human lung cancer cell lines.

BACKGROUND: Lung cancer, a chronic and heterogeneous disease, is the leading cause of cancer-related death on a global scale. Presently, despite a variety of available treatments, their effectiveness is limited, often resulting in considerable toxicity and adverse effects. Additionally, the development of chemoresistance in cancer cells poses a challenge. Trilobolide-6-O-isobutyrate (TBB), a natural sesquiterpene lactone extracted from Sphagneticola trilobata, has exhibited antitumor effects. Its pharmacological properties in NSCLC lung cancer, however, have not been explored. PURPOSE: This study evaluated the impact of TBB on the A549 and NCI-H460 tumor cell lines in vitro, examining its antiproliferative properties and initial mechanisms of cell death. METHODS: TBB, obtained at 98 % purity from S. trilobata leaves, was characterized using chromatographic techniques. Subsequently, its impact on inhibiting tumor cell proliferation in vitro, TBB-induced cytotoxicity in LLC-MK2, THP-1, AMJ2-C11 cells, as well as its effects on sheep erythrocytes, and the underlying mechanisms of cell death, were assessed. RESULTS: In silico predictions have shown promising drug-likeness potential for TBB, indicating high oral bioavailability and intestinal absorption. Treatment of A549 and NCI-H460 human tumor cells with TBB demonstrated a direct impact, inducing significant morphological and structural alterations. TBB also reduced migratory capacity without causing toxicity at lower concentrations to LLC-MK2, THP-1 and AMJ2-C11 cell lines. This antiproliferative effect correlated with elevated oxidative stress, characterized by increased levels of ROS, superoxide anion radicals and NO, accompanied by a decrease in antioxidant markers: SOD and GSH. TBB-stress-induced led to changes in cell metabolism, fostering the accumulation of lipid droplets and autophagic vacuoles. Stress also resulted in compromised mitochondrial integrity, a crucial aspect of cellular function. Additionally, TBB prompted apoptosis-like cell death through activation of caspase 3/7 stressors. CONCLUSION: These findings underscore the potential of TBB as a promising candidate for future studies and suggest its viability as an additional component in the development of novel anticancer drugs prototypes.

Doxorubicin-Loaded Iron Oxide Nanoparticles Induce Oxidative Stress and Cell Cycle Arrest in Breast Cancer Cells.

Cancer is one of the most common diseases nowadays and derives from the uncontrollable growth of a single cell. Magnetic nanoparticles (NpMag) offer various possibilities for use in the biomedical area, including drug delivery mediated by magnetic fields. In the current study, we evaluated the in vitro effects of iron-oxide magnetic nanoparticles conjugated with the antitumor drug doxorubicin (Dox) on human breast cancer cells. Our results revealed that magnetic nanoparticles with Dox (NpMag+Dox) induce cellular redox imbalance in MCF-7 cells. We also demonstrate that iron-oxide nanoparticles functionalized with Dox induce oxidative stress evidenced by DNA damage, lipid peroxidation, cell membrane disruption, and loss of mitochondria potential. As a result, NpMag+Dox drives MCF-7 cells to stop the cell cycle and decrease cell migration. The association of NpMg+Dox induced a better delivery of Dox to MCF cells, mainly in the presence of a magnetic field, increasing the death of MCF cells which might reduce the toxicity for healthy cells providing a better efficacy for the treatment. Thus, iron-oxide nanoparticles and doxorubicin conjugated may be candidate for anticancer therapy.

Polycaprolactone nanofibers colonized by HeLa cells: an alternative for in vitro investigation of fungal infection.

Introduction: In vitro 3D equivalent tissues can be used for studies of fungal infections. Objectives: To develop 3D electrospun nanofibers using polycaprolactone (PCL) colonized by HeLa cells as a possible in vitro model for the investigation of fungal infection. Materials & methods: A PCL solution was synthesized and electrospun. HeLa cells were cultured on the nanostructured PCL scaffolds, forming a 3D structure. Physicochemical, biological and Candida albicans infection assays were performed in this model. Results: The nanostructured PCL scaffolds showed favorable physicochemical characteristics and allowed the colonization of HeLa cells, which showed indications of extracellular matrix production. Conclusions: Fungal infection was evidenced in the 3D nanostructured PCL scaffolds, being viable, economical and compatible to study fungal infections in vitro.

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.

Synergistic Antileishmanial Effect of Oregano Essential Oil and Silver Nanoparticles: Mechanisms of Action on Leishmania amazonensis.

American tegumentary leishmaniasis, a zoonotic disease caused by the Leishmania genus, poses significant challenges in treatment, including administration difficulty, low efficacy, and parasite resistance. Novel compounds or associations offer alternative therapies, and natural products such as oregano essential oil (OEO), extracted from Origanum vulgare, have been extensively researched due to biological effects, including antibacterial, antifungal, and antiparasitic properties. Silver nanoparticles (AgNp), a nanomaterial with compelling antimicrobial and antiparasitic activity, have been shown to exhibit potent leishmanicidal properties. We evaluated the in vitro effect of OEO and AgNp-Bio association on L. amazonensis and the death mechanisms of the parasite involved. Our results demonstrated a synergistic antileishmanial effect of OEO + AgNp on promastigote forms and L. amazonensis-infected macrophages, which induced morphological and ultrastructural changes in promastigotes. Subsequently, we investigated the mechanisms underlying parasite death and showed an increase in NO, ROS, mitochondrial depolarization, accumulation of lipid-storage bodies, autophagic vacuoles, phosphatidylserine exposure, and damage to the plasma membrane. Moreover, the association resulted in a reduction in the percentage of infected cells and the number of amastigotes per macrophage. In conclusion, our findings establish that OEO + AgNp elicits a late apoptosis-like mechanism to combat promastigote forms and promotes ROS and NO production in infected macrophages to target intracellular amastigote forms.

Trypanocidal action of (-)-elatol involves an oxidative stress triggered by mitochondria dysfunction.

Natural compounds have shown good potential for the discovery of new chemotherapeutics for the treatment of Chagas' disease. Recently, our group reported the effective trypanocidal activity of (-)-elatol, extracted from the red macroalgae Laurencia dendroidea present in the Brazilian coast against Trypanosoma cruzi. However, the mechanism of action of this compound has remained unclear. There are only hypotheses concerning its action on mitochondrial function. Here, we further investigated the mechanisms of action of (-)-elatol on trypomastigotes of T. cruzi. For this, we evaluated some biochemical alterations in trypomastigotes treated with (-)-elatol. Our results show that (-)-elatol induced depolarization of the mitochondrial membrane, an increase in the formation of mitochondrial superoxide anion and loss of cell membrane and DNA integrity. Additionally, (-)-elatol induced formation of autophagic vacuoles and a decrease in cell volume. All together, these results suggest that the trypanocidal action of (-)-elatol involves multiple events and mitochondria might be the initial target organelle. Our hypothesis is that the mitochondrial dysfunction leads to an increase of ROS production through the electron transport chain, which affects cell membrane and DNA integrity leading to different types of parasite death.

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.