Copaifera langsdorffii Oleoresin-Loaded Nanostructured Lipid Carrier Emulgel Improves Cutaneous Healing by Anti-Inflammatory and Re-Epithelialization Mechanisms.

The skin is essential to the integrity of the organism. The disruption of this organ promotes a wound, and the organism starts the healing to reconstruct the skin. Copaifera langsdorffii is a tree used in folk medicine to treat skin affections, with antioxidant and anti-inflammatory properties. In our study, the oleoresin of the plant was associated with nanostructured lipid carriers, aiming to evaluate the healing potential of this formulation and compare the treatment with reference drugs used in wound healing. Male Wistar rats were used to perform the excision wound model, with the macroscopic analysis of wound retraction. Skin samples were used in histological, immunohistochemical, and biochemical analyses. The results showed the wound retraction in the oleoresin-treated group, mediated by α-smooth muscle actin (α-SMA). Biochemical assays revealed the anti-inflammatory mechanism of the oleoresin-treated group, increasing interleukin-10 (IL-10) concentration and decreasing pro-inflammatory cytokines. Histopathological and immunohistochemical results showed the improvement of re-epithelialization and tissue remodeling in the Copaifera langsdorffii group, with an increase in laminin-γ2, a decrease in desmoglein-3 and an increase in collagen remodeling. These findings indicate the wound healing potential of nanostructured lipid carriers associated with Copaifera langsdorffii oleoresin in skin wounds, which can be helpful as a future alternative treatment for skin wounds.

Brazilian red propolis extract free and encapsulated into polymeric nanoparticles against ovarian cancer: formulation, characterisation and biological assays in 2D and 3D models.

Cancer incidence worldwide is alarming and among the cancers that affect women ovarian cancer is the most fatal. Many side effects are associated with conventional therapies and none of them are completely effective, so the development of new treatments is necessary. Brazilian red propolis extract is a natural product with complex composition and great potential for cancer treatment. However, its clinical application is harmed due to unfavourable physicochemical characteristics. To enable its application encapsulation in nanoparticles can be used. OBJECTIVES: The aims of this work were to develop polymeric nanoparticles with Brazilian red propolis extract and compare their action with the free extract against ovarian cancer cells. METHODS: Box Behnken design was used and nanoparticles were characterised using the techniques dynamic light scattering, nanoparticle tracking analysis, transmission electron microscopy, differential scanning calorimetry and encapsulation efficiency. Activity against OVCAR-3 was also tested on 2D and 3D models. KEY FINDINGS: Nanoparticles' sizes were ~200 nm with monomodal size distribution, negative zeta potential, spherical shape and with extract molecularly dispersed. Encapsulation efficiency was above 97% for the biomarkers chosen. Nanoparticles had greater efficacy in comparison with free propolis in OVCAR-3. CONCLUSIONS: So far, the nanoparticles here described have the potential to be a chemotherapy treatment in the future.

Nanoparticle Systems for Cancer Phototherapy: An Overview.

Photodynamic therapy (PDT) and photothermal therapy (PTT) are photo-mediated treatments with different mechanisms of action that can be addressed for cancer treatment. Both phototherapies are highly successful and barely or non-invasive types of treatment that have gained attention in the past few years. The death of cancer cells because of the application of these therapies is caused by the formation of reactive oxygen species, that leads to oxidative stress for the case of photodynamic therapy and the generation of heat for the case of photothermal therapies. The advancement of nanotechnology allowed significant benefit to these therapies using nanoparticles, allowing both tuning of the process and an increase of effectiveness. The encapsulation of drugs, development of the most different organic and inorganic nanoparticles as well as the possibility of surfaces' functionalization are some strategies used to combine phototherapy and nanotechnology, with the aim of an effective treatment with minimal side effects. This article presents an overview on the use of nanostructures in association with phototherapy, in the view of cancer treatment.

Hybrid Nanoparticles as an Efficient Porphyrin Delivery System for Cancer Cells to Enhance Photodynamic Therapy.

Photodynamic therapy (PDT) is a potential non-invasive approach for application in oncological diseases, based on the activation of a photosensitizer (PS) by light at a specific wavelength in the presence of molecular oxygen to produce reactive oxygen species (ROS) that trigger the death tumor cells. In this context, porphyrins are interesting PS because they are robust, have high chemical, photo, thermal, and oxidative stability, and can generate singlet oxygen (1O2). However, porphyrins exhibit low solubility and a strong tendency to aggregate in a biological environment which limits their clinical application. To overcome these challenges, we developed hybrid nanostructures to immobilize 5,10,15,20-tetrakis[(4-carboxyphenyl) thio-2,3,5,6-tetrafluorophenyl] (P), a new third-generation PS. The biological effect of this system was evaluated against bladder cancer (BC) cells with or without light exposition. The nanostructure composed of lipid carriers coated by porphyrin-chitosan (P-HNP), presented a size of ca. 130 nm and low polydispersity (ca. 0.25). The presence of the porphyrin-chitosan (P-chitosan) on lipid nanoparticle surfaces increased the nanoparticle size, changed the zeta potential to positive, decreased the recrystallization index, and increased the thermal stability of nanoparticles. Furthermore, P-chitosan incorporation on nanoparticles increased the stability and enhanced the self-organization of the system and the formation of spherical structures, as observed by small-angle X-ray scattering (SAXS) analysis. Furthermore, the immobilization process maintained the P photoactivity and improved the photophysical properties of PS, minimizing its aggregation in the cell culture medium. In the photoinduction assays, the P-HNP displayed high phototoxicity with IC50 3.2-folds lower than free porphyrin. This higher cytotoxic effect can be correlated to the high cellular uptake of porphyrin immobilized, as observed by confocal images. Moreover, the coated nanoparticles showed mucoadhesive properties interesting to its application in vivo. Therefore, the physical and chemical properties of nanoparticles may be relevant to improve the porphyrin photodynamic activity in BC cells.

Development of Photoprotective Formulations Containing Nanostructured Lipid Carriers: Sun Protection Factor, Physical-Mechanical and Sensorial Properties.

The effects of ultraviolet (UV) radiation emitted by the sun are cumulative and can result in chemical changes such as the generation of reactive oxygen species (ROS), leading to the regular use of sunscreen. As an alternative, the use of antioxidants, such as quercetin, into sunscreen can control these effects and provide additional skin photoprotection. However, quercetin presents low stability and poor permeation, alternatively, the encapsulation in nanoparticles can improve the stability and skin permeation. Thus, this study aimed to develop photoprotective formulations containing nanoencapsulated quercetin, characterize the physical-mechanical and sensorial properties, and evaluate the influence of nanocarriers on sun protection factor (SPF) and the immediate clinical effects. Sunscreen formulations with or without antioxidants in a free form or loaded in nanostructured lipid carriers (NLCs) were developed. After the stability, rheological behavior, texture profile, and in vivo SPF (sun protector factor) evaluation, sixty female participants, aged between 20 and 35 years, were enclosed to evaluate the sensorial properties and immediate clinical effects of the formulation in the skin hydration using biophysical and skin imaging techniques. The correlation of rheological behavior, texture profile, and sensory properties enabled the correct choice of formulation ingredients. In addition, the use of NLCs with quercetin significantly improved the SPF in vivo of the developed photoprotective formulation, without increasing the amount of UV filters. Finally, the association of NLCs in the photoprotective formulation showed synergistic effects in the SPF and an improvement in the skin barrier function and hydration.

Assessing the cytotoxic potential of glycoalkaloidic extract in nanoparticles against bladder cancer cells.

OBJECTIVE: This study proposed to use the nanotechnology to deliver glycoalkaloidic extract (AE) to bladder cancer cells, evaluating their activity in 2D and 3D models and the biological mechanism of cell death. METHODS: NPs were prepared by nanoprecipitation method using polylactic acid (PLA) and characterized considering their size, charge, particle concentration and stability. The cytotoxicity was evaluated in 2D and 3D model, and the apoptosis and cell cycle were investigated using flow cytometry. KEY FINDINGS: NPs loading AE (NP-AE) had diameter around 125 ± 6 nm (PdI <0.1) and negative charge. The encapsulation efficiency of SM and SS was higher than 85% for both compounds. The obtained formulation showed a significant in-vitro cytotoxic effect against RT4 cells in a dose-dependent manner with IC50 two fold lower than the free AE. The cytotoxic effect of NP-AE was mediated by apoptosis and cell cycle arrested in the S phase. RT4 cells cultured under 3D conditions exhibited a higher resistance to the treatments (IC50 ~ three fold higher than in 2D cell culture). CONCLUSION: The NP-AE might be a promising nanocarrier to load and deliver glycoalkaloids against bladder cancer.

Development of nanoparticles from natural lipids for topical delivery of thymol: Investigation of its anti-inflammatory properties.

Wound healing involves the integration of biological and molecular events and, in case of chronic wounds, the use of drugs can be associated to side effects. Therefore, there is a search for alternatives therapeutics that encompass minimal toxicity. The use of natural compounds is an attractive approach for treating inflammatory disorders, wounds and burns. In this context, thymol has antimicrobial, antioxidant and antiseptic properties and is a promising compound in wound healing and inflammation management. However, essential oils and their constituents such as thymol present high volatility and can also easily decompose, thereby the encapsulation of these compounds into nanoparticles may be an efficient approach to modulate the release of the active ingredient, to increase the physical stability and to eventually reduce the toxicity. The aims of this work were to encapsulate thymol in nanostructured lipid carriers (NLCs) composed of natural lipids and assess its in vivo anti-inflammatory and antipsoriatic activity. The carrier containing thymol was produced by sonication method and showed 107.7 (±3.8) nm of size, zeta potential of -11.6 (±2.9) mV and entrapment efficiency of 89.1 (±4.2)%. Thymol-NLCs were incorporated into a gel and the final formulation presented rheological characteristics and pH suitable for topic application. In addition, the gel containing thymol-NLCs was tested in vivo on two different mouse models of skin inflammation, showing anti-inflammatory activity. Finally, this formulation was tested in an imiquimod-induced psoriasis mouse model and showed improved healing, compared to negative control. Therefore, thymol-NLCs is an interesting formulation for future treatment of inflammatory skin diseases.

Comparative Analysis of 3D Bladder Tumor Spheroids Obtained by Forced Floating and Hanging Drop Methods for Drug Screening.

Introduction: Cell-based assays using three-dimensional (3D) cell cultures may reflect the antitumor activity of compounds more accurately, since these models reproduce the tumor microenvironment better. Methods: Here, we report a comparative analysis of cell behavior in the two most widely employed methods for 3D spheroid culture, forced floating (Ultra-low Attachment, ULA, plates), and hanging drop (HD) methods, using the RT4 human bladder cancer cell line as a model. The morphology parameters and growth/metabolism of the spheroids generated were first characterized, using four different cell-seeding concentrations (0.5, 1.25, 2.5, and 3.75 × 104 cells/mL), and then, subjected to drug resistance evaluation. Results: Both methods generated spheroids with a smooth surface and round shape in a spheroidization time of about 48 h, regardless of the cell-seeding concentration used. Reduced cell growth and metabolism was observed in 3D cultures compared to two-dimensional (2D) cultures. The optimal range of spheroid diameter (300-500 µm) was obtained using cultures initiated with 0.5 and 1.25 × 104 cells/mL for the ULA method and 2.5 and 3.75 × 104 cells/mL for the HD method. RT4 cells cultured under 3D conditions also exhibited a higher resistance to doxorubicin (IC50 of 1.00 and 0.83 µg/mL for the ULA and HD methods, respectively) compared to 2D cultures (IC50 ranging from 0.39 to 0.43). Conclusions: Comparing the results, we concluded that the forced floating method using ULA plates was considered more suitable and straightforward to generate RT4 spheroids for drug screening/cytotoxicity assays. The results presented here also contribute to the improvement in the standardization of the 3D cultures required for widespread application.

Polymeric Nanoparticles of Enoxaparin as a Delivery System: In Vivo Evaluation in Normal Rats and in a Venous Thrombosis Rat Model.

Enoxaparin is an anticoagulant widely used in the treatment and prophylaxis of deep vein thrombosis (DVT). The subcutaneous route of administration, sometimes in repeated doses during 24 hours, represents a limitation to its use. Thus, the development of a product that can be administered either subcutaneously, in a smaller number of applications becomes a major challenge, with interesting clinical applications. The use of a system for sustained release of drugs can help to meet that goal, by protecting and enabling a gradual released of the agent. This study consisted of the evaluation of in vivo anticoagulant and antithrombotic activity of biodegradable nanoparticles of poly (ε-caprolactone) (PCL) with enoxaparin after subcutaneous injection. The nanoparticles were prepared by the method of double emulsion (w/o/w) and solvent evaporation. Subcutaneous enoxaparin encapsulated in PCL nanoparticles (1000 IU/kg) showed a sustained release in vivo for up to 12 hours (Cmax 0.62 IU/mL) a significantly longer period (P < 0.01) when compared to free enoxaparin (1000 IU/Kg) that disappeared after 9 hours (Cmax 1.50 IU/mL), however with lower anti-Xa activity. The antithrombotic action of enoxaparin-nanoparticles was tested in a DVT model by stasis in rats. There were virtually no formation of venous thrombosis in any of the rats that received enoxaparin encapsulated in nanoparticles (0.03 mg), with a significant difference when compared to groups that received saline (17.2 mg, P < 0.001) and free enoxaparin (2.87 mg, P = 0.001). In summary, enoxaparin-encapsulated in polymeric nanoparticles showed a sustained release for a greater period than that of enoxaparin, and with excellent antithrombotic action. These results corroborate the promising use of pharmacological nanoparticles in clinical practice.

Preparation of an agar-silver nanoparticles (A-AgNp) film for increasing the shelf-life of fruits.

Preparation of protective coating possessing antimicrobial properties is present day need as they increase the shelf life of fruits and vegetables. In the present study, preparation of agar-silver nanoparticle film for increasing the shelf life of fruits is reported. Silver nanoparticles (Ag-NPs) biosynthesised using an extract of Ocimum sanctum leaves, were mixed with agar-agar to prepare an agar-silver nanoparticles (A-AgNp) film. This film was surface-coated over the fruits, Citrus aurantifolium (Thornless lime) and Pyrus malus (Apple), and evaluated for the determination of antimicrobial activity of A-AgNp films using disc diffusion method, weight loss and shelf life of fruits. This study demonstrates that these A-AgNp films possess antimicrobial activity and also increase the shelf life of fruits.

Biogenic antimicrobial silver nanoparticles produced by fungi.

Aspergillus tubingensis and Bionectria ochroleuca showed excellent extracellular ability to synthesize silver nanoparticles (Ag NP), spherical in shape and 35 ± 10 nm in size. Ag NP were characterized by transmission electron microscopy, X-ray diffraction analysis, and photon correlation spectroscopy for particle size and zeta potential. Proteins present in the fungal filtrate and in Ag NP dispersion were analyzed by electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Ag NP showed pronounced antifungal activity against Candida sp, frequently occurring in hospital infections, with minimal inhibitory concentration in the range of 0.11-1.75 µg/mL. Regarding antibacterial activity, nanoparticles produced by A. tubingensis were more effective compared to the other fungus, inhibiting 98.0 % of Pseudomonas. aeruginosa growth at 0.28 µg/mL. A. tubingensis synthesized Ag NP with surprisingly high and positive surface potential, differing greatly from all known fungi. These data open the possibility of obtaining biogenic Ag NP with positive surface potential and new applications.

Chitosan-solid lipid nanoparticles as carriers for topical delivery of tretinoin.

Tretinoin (TRE) or all-trans retinoic acid is employed in the topical treatment of various skin diseases including acne and psoriasis. However, its use is strongly limited by side effects and high chemical instability. TRE encapsulation in nanostructured systems reduces these problems. Chitosan is a biopolymer that exhibits a number of interesting properties such as bioadhesion and antibacterial activity. The aim of this work was to prepare and characterize solid lipid nanoparticles (SLN) containing TRE, with and without addition of chitosan, to assess their in vitro cytotoxicity in keratinocytes and to evaluate their antibacterial activity against bacteria related to acne. SLN without (SLN-TRE) and with (SLN-chitosan-TRE) chitosan were prepared by hot high pressure homogenization. The hydrodynamic mean diameter and zeta potential were 162.7±1.4 nm and -31.9±2.0 mV for SLN-TRE, and 284.8±15.0 nm and 55.9±3.1 mV for SLN-chitosan-TRE. The SLN-chitosan-TRE exhibited high encapsulation efficiency, high physical stability in the tested period (one year), were not cytotoxic to keratinocytes and showed high antibacterial activity against P. acnes and S. aureus. Therefore chitosan-SLN can be good candidates to encapsulate TRE and to increase its therapeutic efficacy in the topical treatment of acne.

Mechanistic aspects in the biogenic synthesis of extracellular metal nanoparticles by peptides, bacteria, fungi, and plants.

Metal nanoparticles have been studied and applied in many areas including the biomedical, agricultural, electronic fields, etc. Several products of colloidal silver are already on the market. Research on new, eco-friendly and cheaper methods has been initiated. Biological production of metal nanoparticles has been studied by many researchers due to the convenience of the method that produces small particles stabilized by protein. However, the mechanism involved in this production has not yet been elucidated although hypothetical mechanisms have been proposed in the literature. Thus, this review discusses the various mechanisms provided for the biological synthesis of metal nanoparticles by peptides, bacteria, fungi, and plants. One thing that is clear is that the mechanistic aspects in some of the biological systems need more detailed studies.

Nanocytotoxicity: violacein and violacein-loaded poly (D, L-lactide-co-glycolide) nanoparticles acting on human leukemic cells.

Violacein is a compound obtained from Chromobacterium violaceum, a bacterium found in the Amazonian region. Violacein-loaded poly (D, L-lactide-co-glycolide) nanoparticles has a similar inhibitory effect evaluated by trypan blue assay on leukemic HL60 cells than the free form. However, the cytotoxic effects evaluated by phosphatase activity and MTT reduction assays were lower for the encapsulated form than for free violacein. Based on morphological changes, violacein and violacein entrapped in nanoparticles were found to induce terminal differentiation (assessed by nitro blue tetrazolium reduction) in HL60 cells. Thus, both formulations inhibit HL60 cell growth in vitro, partly by inducing cytotoxic effects and cell differentiation. Flow cytometric analysis of HL60 cells after treatment for 12 h showed that violacein-loaded PLGA induced apoptosis, with maximum cell death at a concentration of 2 microM. Violacein and violacein/PLGA induced opposite effects on the mitochondrial swelling which indicates altered mitochondrial function. The mitochondrial activity was also checked by flow cytometry studies. Labelled cells with the probe JC1 displayed a basal hypopolarized status of the mitochondria in treated cells. Based on morphological changes, alterations in phospholipid asymmetry and changes in mitochondrial polarization, violacein and nanoparticles containing violacein were found to trigger cell death by apoptosis. These methodologies are promising as diagnostic and mechanistic effects of nanoparticles in cell cultures.

New aspects of nanopharmaceutical delivery systems.

Nanobiotechnology, involving biological systems manufactured at the molecular level, is a multidisciplinary field that has fostered the development of nanoscaled pharmaceutical delivery devices. Micelles, liposomes, solid lipid nanoparticles, polymeric nanoparticles, functionalized nanoparticles, nanocrystals, cyclodextrins, dendrimers, nanotubes and metallic nanoparticles have been used as strategies to deliver conventional pharmaceuticals or substances such as peptides, recombinant proteins, vaccines and nucleotides. Nanoparticles and other colloidal pharmaceutical delivery systems modify many physicochemical properties, thus resulting in changes in the body distribution and other pharmacological processes. These changes can lead to pharmaceutical delivery at specific sites and reduce side effects. Therefore, nanoparticles can improve the therapeutic efficiency, being excellent carriers for biological molecules, including enzymes, recombinant proteins and nucleic acid. This review discusses different pharmaceutical carrier systems, and their potential and limitations in the field of pharmaceutical technology. Products with these technologies which have been approved by the FDA in different clinical phases and which are on the market will be also discussed.

Mechanistic aspects of biosynthesis of silver nanoparticles by several Fusarium oxysporum strains.

Extracellular production of metal nanoparticles by several strains of the fungus Fusarium oxysporum was carried out. It was found that aqueous silver ions when exposed to several Fusarium oxysporum strains are reduced in solution, thereby leading to the formation of silver hydrosol. The silver nanoparticles were in the range of 20-50 nm in dimensions. The reduction of the metal ions occurs by a nitrate-dependent reductase and a shuttle quinone extracellular process. The potentialities of this nanotechnological design based in fugal biosynthesis of nanoparticles for several technical applications are important, including their high potential as antibacterial material.