Current Applications of Plant-Based Drug Delivery Nano Systems for Leishmaniasis Treatment.

Leishmania is a trypanosomatid that causes leishmaniasis. It is transmitted to vertebrate hosts during the blood meal of phlebotomine sandflies. The clinical manifestations of the disease are associated with several factors, such as the Leishmania species, virulence and pathogenicity, the host-parasite relationship, and the host's immune system. Although its causative agents have been known and studied for decades, there have been few advances in the chemotherapy of leishmaniasis. The urgency of more selective and less toxic alternatives for the treatment of leishmaniasis leads to research focused on the study of new pharmaceuticals, improvement of existing drugs, and new routes of drug administration. Natural resources of plant origin are promising sources of bioactive substances, and the use of ethnopharmacology and folk medicine leads to interest in studying new medications from phytocomplexes. However, the intrinsic low water solubility of plant derivatives is an obstacle to developing a therapeutic product. Nanotechnology could help overcome these obstacles by improving the availability of common substances in water. To contribute to this scenario, this article provides a review of nanocarriers developed for delivering plant-extracted compounds to treat clinical forms of leishmaniasis and critically analyzing them and pointing out the future perspectives for their application.

In Vitro and In Vivo Effect of pH-Sensitive PLGA-TPGS-Based Hybrid Nanoparticles Loaded with Doxorubicin for Breast Cancer Therapy.

Doxorubicin (DOX) is an antineoplastic agent clinically employed for treating breast cancer patients. Despite its effectiveness, its inherent adverse toxic side effects often limit its clinical application. To overcome these drawbacks, lipid-polymer hybrid nanoparticles (LPNP) arise as promising nanoplatforms that combine the advantages of both liposomes and polymeric nanoparticles into a single delivery system. Alpha-tocopherol succinate (TS) is a derivative of vitamin E that shows potent anticancer mechanisms, and it is an interesting approach as adjuvant. In this study, we designed a pH-sensitive PLGA-polymer-core/TPGS-lipid-shell hybrid nanoparticle, loaded with DOX and TS (LPNP_TS-DOX). Nanoparticles were physicochemically and morphologically characterized. Cytotoxicity studies, migration assay, and cellular uptake were performed in 4T1, MCF-7, and MDA-MB-231 cell lines. Antitumor activity in vivo was evaluated in 4T1 breast tumor-bearing mice. In vitro studies showed a significant reduction in cell viability, cell migration, and an increase in cellular uptake for the 4T1 cell line compared to free DOX. In vivo antitumor activity showed that LPNP-TS-DOX was more effective in controlling tumor growth than other treatments. The high cellular internalization and the pH-triggered payload release of DOX lead to the increased accumulation of the drugs in the tumor area, along with the synergic combination with TS, culminating in greater antitumor efficacy. These data support LPNP-TS-DOX as a promising drug delivery system for breast cancer treatment.

Formulation of Amphotericin B in PEGylated Liposomes for Improved Treatment of Cutaneous Leishmaniasis by Parenteral and Oral Routes.

Liposomal amphotericin B (AmB) or AmBisome® is the most effective and safe therapeutic agent for visceral leishmaniasis (VL), but its clinical efficacy is limited in cutaneous leishmaniasis (CL) and HIV/VL co-infection. The aim of this work was to develop a formulation of AmB in PEGylated liposomes and compare its efficacy to AmBisome® in a murine model of CL. Formulations of AmB in conventional and PEGylated liposomes were characterized for particle size and morphology, drug encapsulation efficiency and aggregation state. Those were compared to AmBisome® in Leishmania amazonensis-infected BALB/c mice for their effects on the lesion size growth and parasite load. The conventional and PEGylated formulations showed vesicles with 100-130 nm diameter and low polydispersity, incorporating more than 95% of AmB under the non-aggregated form. Following parenteral administration in the murine model of CL, the PEGylated formulation of AmB significantly reduced the lesion size growth and parasite load, in comparison to control groups, in contrast to conventional liposomal AmB. The PEGylated formulation of AmB was also effective when given by oral route on a 2-day regimen. This work reports for the first time that PEGylated liposomal AmB can improve the treatment of experimental cutaneous leishmaniasis by both parenteral and oral routes.

Memantine-Derived Schiff Bases as Transdermal Prodrug Candidates.

Condensation reactions of salicylaldehyde, 2-pyridinecarboxaldehyde, and pyridoxaldehyde with memantine (Me) produced novel memantine-derived Schiff bases (1-3). Speciation predictions and calculations of Log P, Log D, and of the percentage (%) of neutral species for (1-3) were carried out. In comparison with Me, the Schiff bases presented increased log P and log D in all cases and pH values, suggesting higher hydrophobicity. The determined solubilities in n-octanol were 34.7 mg/mL for memantine hydrochloride and 67.3 mg/mL for (3). According to the molecular weights and calculated logP, compounds (1-3) are suitable for transdermal administration, especially compound (3). In addition, hydrolysis of 3 with the release of pyridoxal, a daily cofactor in human metabolism, was observed. The results suggested that 3 is the most promising compound and that formation of the pyridoxal Schiff base with Me might be an effective strategy to obtain a prodrug candidate with increased lipophilicity, which would be able to passively cross biological barriers during transdermal delivery and might have applications in the treatment of Alzheimer's disease and other neurological disorders.

Stepwise Protocols for Preparation and Use of Porcine Ear Skin for in Vitro Skin Permeation Studies Using Franz Diffusion Cells.

The skin, the largest organ of the body, is an attractive route of topical and systemic drug administration. During the development of topical formulations, in vitro skin permeation studies using biological membranes mounted in Franz diffusion cells are a useful tool to assess the permeation of substances through the skin, and are recommended by the Organization for Economic Cooperation and Development (OECD). Among the types of biological membranes used in such studies, porcine ear skin has been identified as the most promising, due to its similarities to human skin and its greater accessibility as compared to human skin. To standardize techniques for the preparation and use of porcine ear skin as biological membrane, here we present systematic procedures for the selection of porcine ears, their cleaning, the removal of skin from cartilage, its transformation into membranes, and its use for the in vitro assessment of the permeation of drugs from topical formulations. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Obtaining porcine ear membranes Basic Protocol 2: Preparation of membranes from porcine ear skin and use of membranes for in vitro skin permeation studies.

Liposomal Amphotericin B for Treatment of Leishmaniasis: From the Identification of Critical Physicochemical Attributes to the Design of Effective Topical and Oral Formulations.

The liposomal amphotericin B (AmB) formulation, AmBisome®, still represents the best therapeutic option for cutaneous and visceral leishmaniasis. However, its clinical efficacy depends on the patient's immunological status, the clinical manifestation and the endemic region. Moreover, the need for parenteral administration, its side effects and high cost significantly limit its use in developing countries. This review reports the progress achieved thus far toward the understanding of the mechanism responsible for the reduced toxicity of liposomal AmB formulations and the factors that influence their efficacy against leishmaniasis. It also presents the recent advances in the development of more effective liposomal AmB formulations, including topical and oral liposome formulations. The critical role of the AmB aggregation state and release rate in the reduction of drug toxicity and in the drug efficacy by non-invasive routes is emphasized. This paper is expected to guide future research and development of innovative liposomal formulations of AmB.

Nanoencapsulated Doxorubicin Prevents Mucositis Development in Mice.

Doxorubicin (DOX), a chemotherapy drug successfully used in the therapy of various types of cancer, is currently associated with the mucositis development, an inflammation that can cause ulcerative lesions in the mucosa of the gastrointestinal tract, abdominal pain and secondary infections. To increase the safety of the chemotherapy, we loaded DOX into nanostructured lipid carriers (NLCs). The NLC-DOX was characterized by HPLC, DLS, NTA, Zeta potential, FTIR, DSC, TEM and cryogenic-TEM. The ability of NLC-DOX to control the DOX release was evaluated through in vitro release studies. Moreover, the effect of NLC-DOX on intestinal mucosa was compared to a free DOX solution in C57BL/6 mice. The NLC-DOX showed spherical shape, high drug encapsulation efficiency (84.8 ± 4.6%), high drug loading (55.2 ± 3.4 mg/g) and low average diameter (66.0-78.8 nm). The DSC and FTIR analyses showed high interaction between the NLC components, resulting in controlled drug release. Treatment with NLC-DOX attenuated DOX-induced mucositis in mice, improving shortening on villus height and crypt depth, decreased inflammatory parameters, preserved intestinal permeability and increased expression of tight junctions (ZO-1 and Ocludin). These results indicated that encapsulation of DOX in NLCs is viable and reduces the drug toxicity to mucosal structures.

Efficacy of nanoemulsion with Pterodon emarginatus Vogel oleoresin for topical treatment of cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is a neglected tropical skin disease caused by the protozoan genus Leishmania. The treatment is restricted to a handful number of drugs that exhibit toxic effects, limited efficacy, and drug resistance. Additionally, developing an effective topical treatment is still an enormous unmet medical challenge. Natural oils, e.g. the oleoresin from P. emarginatus fruits (SO), contain various bioactive molecules, especially terpenoid compounds such as diterpenes and sesquiterpenes. However, its use in topical formulations can be impaired due to the natural barrier of the skin for low water solubility compounds. Nanoemulsions (NE) are drug delivery systems able to increase penetration of lipophilic compounds throughout the skin, improving their topical effect. In this context, we propose the use of SO-containing NE (SO-NE) for CL treatment. The SO-NE was produced by a low energy method and presented suitable physicochemical characteristic: average diameter and polydispersity index lower than 180 nm and 0.2, respectively. Leishmania (Leishmania) amazonensis-infected BALB/c mice were given topical doses of SO or SO-NE. The topical use of a combination of SO-NE and intraperitoneal meglumine antimoniate reduced lesion size by 41 % and tissue regeneration was proven by histopathological analyses. In addition, a reduction in the parasitic load and decreased in the level of IFN-γ in the lesion may be associated, as well as a lower level of the cytokine IL-10 may be associated with a less intense inflammatory process. The present study suggests that SO-NE in combination meglumine antimoniate represents a promising alternative for the topical treatment of CL caused by L. (L.) amazonensis.

Ion Pair Strategy in Solid Lipid Nanoparticles: a Targeted Approach to Improve Epidermal Targeting with Controlled Adapalene Release, Resulting Reduced Skin Irritation.

PURPOSE: Adapalene (AD) is one of the main retinoids used in the topical therapy of acne, an extremely common skin disease usually associated with psychological morbidity. However, like other retinoids, AD is frequently associated with skin irritation. To overcome the skin irritation, we proposed the encapsulation of AD in solid lipid nanoparticles (SLNs) using the ion pair strategy. METHODS: The developed SLN-AD was characterized by high-performance liquid chromatography, differential scanning calorimetry, X-ray diffraction, synchrotron small-angle X-ray scattering, and transmission electron microscopy. In vitro permeation tests using porcine skin and in vivo mice skin irritation test were performed to evaluate, respectively, the drug's skin distribution and the skin irritation. RESULTS: The characterization studies were able to demonstrate that the proposed strategy effectively provided high AD encapsulation in SLNs and its incorporation into a hydrophilic gel. Sustained release, epidermal targeting, and less skin irritation were observed for SLN-AD gel in comparison to the marketed AD gel. CONCLUSIONS: The studies demonstrated that the encapsulation of AD in SLNs through the formation of an ion pair is a valuable alternative to diminish the adverse skin reactions caused by AD and can optimize patient adherence to treatment.

Retinoic acid-loaded solid lipid nanoparticles surrounded by chitosan film support diabetic wound healing in in vivo study.

Repair of tissue damaged in diabetic wounds is essential to minimize the cases of amputation of the limbs in millions of diabetic people around the world. Although the all-trans retinoic acid (ATRA) is described as a potential wound healing agent, however its effects are controversial due to adverse reactions that may impair the wound healing during the treatment schedules. Our aim was to design and characterize an ATRA-loaded solid lipid nanoparticles surrounded by chitosan film to promote an ATRA controlled release and to evaluate its effectiveness in promoting wound healing in a diabetic mouse model. The SLN-ATRA were developed using biocompatible lipids without using organic solvent. The SLN-ATRA had high drug entrapment efficiency (98.0 %) and low polydispersity index (PDI) and average diameter, respectively, 0.24 ± 0.02 and 83.0 ± 6 nm. The transmission electron microscope (TEM) image presented that the SLN-ATRA were homogeneous in size and had spherical structures. The incorporation of SLN-ATRA in the chitosan films propitiated a homogeneous distribution of the drug and a controlled drug release. Furthermore, in vivo assay proved that chitosan films containing SLN-ATRA accelerated the closure of wounds of diabetic mice when compared to the control chitosan films without ATRA. SLN-ATRA chitosan films also reduced leukocyte infiltrate in the wound bed, improved collagen deposition, and reduced scar tissue. No sign of skin irritation was observed. These results indicated that SLN-ATRA surrounded in chitosan films are a promising candidate to treat diabetic wounds, improving tissue healing.

α- Tocopherol succinate loaded nano-structed lipid carriers improves antitumor activity of doxorubicin in breast cancer models in vivo.

Combination-based chemotherapies have been the standard treatment for multiple solid tumors since the 1960s. Combined therapies where both agents have toxicity results in dose-limiting effects. α- tocopherol succinate (TS) is an analogue of vitamin E that exhibits antitumor properties in the absence of toxicity. Hence, its combination with a frontline chemotherapy, doxorubicin (DOX) is an alternative to increase antitumor efficacy. Therefore, the aim of this work was to evaluate the antitumor activity of nanostructed lipid carriers (NLC) loaded with TS and DOX. The NLC-TS-DOX were prepared, characterized and radiolabeled with technetium-99m. Cytotoxicity studies were performed in vitro, using two breast cancer cell lines, MDA-MB-231 and 4T1. Biodistribution and antitumor activity were evaluated in 4T1 tumor-bearing mice. The results showed that NLC-TS-DOX had a small diameter (85 nm) and a long blood clearance (T1/2ß = 1107.71 min) that consequently resulted in a higher tumor uptake compared to contralateral muscle for up to 48 h. Drug combination studies in MDA-MB-231 and 4T1 cells showed a combination index below 0.8 at ED50-90 for both cell lines. Interestingly, a high synergism was found at ED90. Antitumor activity showed a better control of tumor growth for animals treated with NLC-ST-DOX. The small particle size, along with the EPR effect and the controlled release of DOX from the particle, associated with the synergic combination between TS and DOX led to an increase of the antitumor efficacy. Therefore, NLC-TS-DOX can be considered a plausible alternative to improve antitumor efficacy in DOX therapeutic regimens.

Nanostructured Lipid Carrier Co-loaded with Doxorubicin and Docosahexaenoic Acid as a Theranostic Agent: Evaluation of Biodistribution and Antitumor Activity in Experimental Model.

PURPOSE: Nanotheranostic platforms, i.e., the combination of both therapeutic and diagnostic agents on a single platform, are emerging as an interesting tool for the personalized cancer medicine. Therefore, the aim of this work was to evaluate the in vivo properties of a Tc-99m-labeled nanostructured lipid carrier (NLC) formulation, co-loaded with doxorubicin (DOX) and docosahexaenoic acid (DHA), for theranostic applications. PROCEDURES: NLC-DHA-DOX were prepared busing the hot melting homogenization method using an emulsification-ultrasound and were radiolabeled with Tc-99m. Biodistribution studies, scintigraphic images, and antitumor activity were performed in 4T1 tumor-bearing mice. RESULTS: NCL was successfully radiolabeled with Tc-99m. Blood clearance showed a relatively long half-life, with blood levels decaying in a biphasic manner (T1/2 α = 38.7 min; T1/2 ß = 516.5 min). The biodistribution profile and scintigraphic images showed higher tumor uptake compared to contralateral muscle in all time-points investigated. Antitumor activity studies showed a substantial tumor growth inhibition ratio for NLC-DHA-DOX formulation. In addition, the formulation showed more favorable toxicity profiles when compared to equivalent doses of free administered drugs, being able to reduce heart and liver damage. CONCLUSIONS: Therefore, NLC-DHA-DOX formulation demonstrated feasibility in breast cancer treatment and diagnosis/monitoring, leading to a new possibility of a theranostic platform.

Doxorubicin-loaded nanocarriers: A comparative study of liposome and nanostructured lipid carrier as alternatives for cancer therapy.

Nowadays cancer is one of the most common causes of deaths worldwide. Conventional antitumor agents still present various problems related to specificity for tumor cells often leading to therapeutic failure. Nanoscale particles are considered potential alternative to direct access of drugs into tumor cells, therefore increasing the drug accumulation and performance. The aim of this study was to evaluate the antitumor activity of doxorubicin (DOX)-loaded nanostructured lipid carriers (NLC) versus liposomes against a breast cancer animal experimental model. NLC-DOX and liposomes-DOX were successfully prepared and characterized. Tumor-bearing mice were divided into five groups (blank-NLC, blank-liposome, DOX, NLC-DOX, liposome-DOX). Each animal received by the tail vein four doses of antitumoral drugs (total dose, 16mg/kg), every 3 days. Antitumor efficacy was assessed by measuring 1) tumor volume, calculating the inhibitory ratio (TV-IR, see after) and 2) acquiring scintigraphic images of the tumor using doxorubicin radiolabeled with technetium-99m as an imaging tumor probe. Liposome-DOX and free DOX did not showed differences in the tumor mean volume, whereas NLC-DOX proved to be the best treatments in controlling the tumor growth. NLC-DOX showed an inhibition ration (TV-IR) of 73.5% while free DOX and liposome-DOX decreased TV-RI of 48.8% and 68.0%, respectively. Tumor was clearly visualized in controls, DOX, and liposome-DOX groups. Yet, regarding the NLC-DOX group, tumor was barely identified by the image, indicating antitumor efficacy. Moreover, both NLC and liposomes proved to be able to delay the occurrence of lung metastasis. In conclusion, results of this study indicated that NLC-DOX might be an alternative strategy to achieve an efficient antitumor activity.

Solid lipid nanoparticles co-loaded with doxorubicin and α-tocopherol succinate are effective against drug-resistant cancer cells in monolayer and 3-D spheroid cancer cell models.

This work aimed to develop solid lipid nanoparticles (SLN) co-loaded with doxorubicin and α-tocopherol succinate (TS) and to evaluate its potential to overcome drug resistance and to increase antitumoral effect in MCF-7/Adr and NCI/Adr cancer cell lines. The SLN were prepared by a hot homogenization method and characterized for size, zeta potential, entrapment efficiency (EE), and drug loading (DL). The cytotoxicity of SLN or penetration was evaluated in MCF-7/Adr and NCI/adr as a monolayer or spheroid cancer cell model. The SLN showed a size in the range of 74-80nm, negative zeta potential, EE of 99%, and DL of 67mg/g. The SLN co-loaded with Dox and TS showed a stronger cytotoxicity against MCF-7/Adr and NCI/Adr cells. In the monolayer model, the doxorubicin co-localization as a free and encapsulated form was higher for the encapsulated drug in MCF-7/Adr and NCI/adr, suggesting a bypassing of P-glycoprotein bomb efflux. For cancer cell spheroids, the SLN co-loaded with doxorubicin and TS showed a prominent cytotoxicity and a greater penetration of doxorubicin.

α-Tocopherol succinate improves encapsulation and anticancer activity of doxorubicin loaded in solid lipid nanoparticles.

This work aimed to develop solid lipid nanoparticles (SLN) co-loaded with doxorubicin and α-tocopheryl succinate (TS), a succinic acid ester of α-tocopherol that exhibits anticancer actions, evaluating the influence of TS on drug encapsulation efficiency. The SLN were characterized for size, zeta potential, entrapment efficiency (EE), and drug release. Studies of in vitro anticancer activity were also conducted. The EE was significantly improved from 30 ± 1% to 96 ± 2% for SLN without and with TS at 0.4%, respectively. In contrast, a reduction in particle size from 298 ± 1 to 79 ± 1 nm was observed for SLN without and with TS respectively. The doxorubicin release data show that SLN provide a controlled drug release. The in vitro studies showed higher cytotoxicity for doxorubicin-TS-loaded SLN than for free doxorubicin in breast cancer cells. These findings suggest that TS-doxorubicin-loaded SLN is a promising alternative for the treatment of cancer.

Metabolomics as a tool to evaluate the toxicity of formulations containing amphotericin B, an antileishmanial drug.

Amphotericin B (AmB) is a drug of choice against life-threatening systemic fungal infections and an alternative therapy for the treatment of all forms of leishmaniasis. It is known that AmB and its conventional formulation cause renal damage; however, the lipid formulations can reduce these effects. The aim of the present study was to identify metabolic changes in mice treated with two different AmB formulations, a nanoemulsion (NE) (lipid system carrier) loaded with AmB and the conventional formulation (C-AmB). For this purpose, metabolic fingerprinting represents a valuable strategy to monitor, in a non-targeted manner, the changes that are at the base of the toxicity mechanism of AmB. Plasma samples of BALB-c mice were collected after treatment with 3 alternate doses of AmB at 1 mg kg-1 administered intravenously and analysed with CE, LC and GC coupled to MS. Blood urea nitrogen (BUN) and plasma creatinine levels were also analysed. Kidney tissue specimens were collected and evaluated. It was not observed that there were any alterations in BUN and creatinine levels as well as in histopathological analysis. Approximately 30 metabolites were identified as potentially related to early C-AmB-induced nephrotoxicity. Disturbances in the arachidonic acid, glycerophospholipid, acylcarnitine and polyunsaturated fatty acid (PUFA) pathways were observed in C-AmB-treated mice. In the AmB-loaded NE group, it was observed that there were fewer metabolic changes, including changes in the plasma levels of cortisol and pyranose. The candidate biomarkers revealed in this study could be useful in the detection of the onset and severity of kidney injury induced by AmB formulations.

Technetium-99m-labeled doxorubicin as an imaging probe for murine breast tumor (4T1 cell line) identification.

OBJECTIVE: Early diagnosis of malignant tumors is essential to successfully plan a radical and curative approach. In this study we describe the direct radiolabeling of doxorubicin (DOX) at physiological pH to identify murine breast tumor (4T1 cells)-bearing BALB/c mice. MATERIALS AND METHODS: Technetium-99m (99mTc) DOX was prepared by adding 99mTc-pertechnetate to a PBS (pH 7.4) solution containing DOX in the presence of stannous chloride. Radiochemical purity and in-vitro stability were determined. The circulation time of 99mTc-DOX was determined by measuring blood radioactivity in healthy animals. Scintigraphic images and biodistribution studies were carried out in tumor-bearing mice at 1, 4, and 8 h after injection. RESULTS: The 99mTc-DOX complex showed high radiochemical purity (99.27 ± 0.34%) and in-vitro stability until 8 h. Tc-DOX levels in blood declined in a biphasic manner, with an α half-life of 4.5 min and a ß half-life of 277.2 min. High uptake was achieved in kidneys, liver, and spleen, because of the drug elimination routes. Moreover, tumor uptake was higher than that of control tissue, resulting in high tumor-to-muscle ratios. CONCLUSION: DOX was successfully labeled with 99mTc-pertechnetate and showed high stability. Biodistribution and scintigraphic studies indicated high tumor-to-muscle ratios in breast tumor-bearing BALB/c mice. These results suggested the feasibility of 99mTc-DOX as a functional agent in tumor diagnosis.

Improved pharmacokinetics and enhanced tumor growth inhibition using a nanostructured lipid carrier loaded with doxorubicin and modified with a layer-by-layer polyelectrolyte coating.

A nanostructured lipid carrier (NLC) loaded with doxorubicin (DOX) has been shown to be cytotoxic against the human cancer cell lines A549 and MCF-7/Adr. In attempts to improve formulation characteristics, enhance pharmacokinetics and antitumor effects, we modified the surface of these NLC with an alternating layer-by-layer (LbL) assembly of polycation and polyanion polyelectrolytes and an additional coating with PEG using a simple method of core shell attachment. The formulation had a narrow size distribution, longer residence in the blood, lower accumulation in the liver, higher accumulation in tumors and a significant tumor growth inhibition effect. Thus, NLC-DOX nanopreparations complexes modified by LbL coating have the potential to enhance the anticancer effects of DOX against tumors.

Novel nanostructured lipid carrier co-loaded with doxorubicin and docosahexaenoic acid demonstrates enhanced in vitro activity and overcomes drug resistance in MCF-7/Adr cells.

PURPOSE: To develop a nanostructured lipid carrier (NLC) co-loaded with doxorubicin and docosahexaenoic acid (DHA) and to evaluate its potential to overcome drug resistance and to increase antitumoral effect in MCF-7/Adr cancer cell line. METHODS: The NLC was prepared by a hot homogenization method and characterized for size, zeta potential, entrapment efficiency (EE) and drug loading (DL). Drug release was evaluated by dialysis in complete DMEM, and NLC aggregation was assayed in the presence of serum. The cytotoxicity of formulations, doxorubicin uptake or penetration were evaluated in MCF-7 and MCF-7/Adr as monolayer or spheroid models. RESULTS: The formulation had a size of about 80 nm, negative zeta potential, EE of 99%, DL of 31 mg/g, a controlled drug release in DMEM and no particles aggregation in presence of serum. The NLC loaded with doxorubicin and DHA showed the same activity as free drugs against MCF-7 but a stronger activity against MCF-7/Adr cells. In monolayer model, the doxorubicin uptake as free and encapsulated form was similar in MCF-7 but higher for the encapsulated drug in MCF-7/Adr, suggesting a bypassing of P-glycoprotein bomb efflux. For spheroids, the NLC loaded with doxorubicin and DHA showed a prominent cytotoxicity and a greater penetration of doxorubicin. CONCLUSIONS: These findings suggest that the co-encapsulation of doxorubicin and DHA in NLC enhances the cytotoxicity and overcomes the doxorubicin resistance in MCF-7/Adr.

Skin-healing activity and toxicological evaluation of a proteinase fraction from Carica candamarcensis.

Previous studies demonstrated that proteinases from latex of C. candamarcensis act as mitogens on fibroblast and epithelial cells and a subsequent report showed their protective, angiogenic and wound healing effects on gastric ulcers. In this study, we present evidence of skin healing activity by the group of proteinases known as P1G10. By using a hairless mouse model, we compared the healing effect following topical application of various concentrations of P1G10. The data confirm that healing actions take place between 0.1 and 1%, without adverse local irritation or systemic toxicological action after a prolonged period of use. The wound healing effect is unaltered when P1G10 is previously inhibited with iodoacetamide. The low permeation of the hydrosoluble formulation Polawax(®) supports the maintenance of the drug at the site of application. These results extend the healing properties of these groups of enzymes in situations of dermatological trauma and open the way to future clinical applications.