Development of liposomes loaded with chloroaluminum phthalocyanine for application of photodynamic therapy in breast cancer.

Chloraluminium phthalocyanine (ClAlPc) has potential therapeutic effect for the treatment of cancer; however, the molecule is lipophilic and may present self-aggregation which limits its clinical success. Thus, nanocarriers like liposomes can improve ClAlPc solubility, reduce off-site toxicity and increase circulation time. For this purpose, developing suitable liposomes requires the evaluation of different lipid compositions. Herein, we aimed to develop liposomes containing soy phosphatidylcholine (SPC), 1,2-distearoyl-sn-glycero- 3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPEPEG2000), cholesterol and oleic acid loaded with ClAlPc using the surface response methodology and the Box-Behnken design. Liposomes with particle size from 110.93 to 374.97 nm and PdI from 0.265 to 0.468 were obtained. The optimized formulation resulted in 69.09% of ClAlPc encapsulated, with particle size and polydispersity index, respectively, at 153.20 nm and 0.309, providing stability and aggregation control. Atomic force microscopy revealed vesicles in a spherical or almost spherical shape, while the analyzes by Differential Scanning Calorimetry (DSC), Powder X-ray Diffraction (PXRD), and Fourier transform infrared spectroscopy (FTIR) suggested that the drug was adequately incorporated into the lipid bilayer of liposomes, in its amorphous state or molecularly dispersed. In vitro studies conducted in breast cancer cells (4T1) showed that liposome improved phototoxicity compared to the ClAlPc solution. ClAlPc-loaded liposomes also enhanced the production of ROS 3-fold compared to the ClAlPc solution. Finally, confocal microscopy and flow cytometry demonstrated the ability of the liposomes to enter cells and deliver the fluorescent ClAlPc photosensitizer with dose and time-dependent effects. Thus, this work showed that Box-Behnken factorial design was an effective strategy for optimizing formulation development. The obtained ClAlPc liposomes can be applied for photodynamic therapy in breast cancer cells.

Resveratrol-Loaded Polymeric Nanoparticles: The Effects of D-α-Tocopheryl Polyethylene Glycol 1000 Succinate (TPGS) on Physicochemical and Biological Properties against Breast Cancer In Vitro and In Vivo.

Resveratrol (RSV), a phytoalexin from grapes and peanuts, has been reported to exhibit antiproliferative effects on various cancer cell lines. In breast cancer, RSV has been demonstrated to exert an antiproliferative effect on both hormone-dependent and hormone-independent breast cancer cell lines. However, RSV is a lipophilic drug, and its therapeutic effect could be improved through nanoencapsulation. Functionalizing polymeric nanoparticles based on polycaprolactone (PCL) with polyethylene glycol 1000 tocopheryl succinate (TPGS) has been reported to prolong drug circulation and reduce drug resistance. However, the effect of TPGS on the physicochemical properties and biological effects of breast cancer cells remains unclear. Therefore, this study aimed to develop RSV-loaded PCL nanoparticles using nanoprecipitation and investigate the effect of TPGS on the nanoparticles' physicochemical characteristics (particle size, zeta potential, encapsulation efficiency, morphology, and release rate) and biological effects on the 4T1 breast cancer cell line (cytotoxicity and cell uptake), in vitro and in vivo. The optimized nanoparticles without TPGS had a size of 138.1 ± 1.8 nm, a polydispersity index (PDI) of 0.182 ± 0.01, a zeta potential of -2.42 ± 0.56 mV, and an encapsulation efficiency of 98.2 ± 0.87%, while nanoparticles with TPGS had a size of 127.5 ± 3.11 nm, PDI of 0.186 ± 0.01, zeta potential of -2.91 ± 0.90 mV, and an encapsulation efficiency of 98.40 ± 0.004%. Scanning electron microscopy revealed spherical nanoparticles with low aggregation tendency. Differential Scanning Calorimetry (DSC) and Fourier Transform Infrared Spectroscopy (FTIR) identified the constituents of the nanoparticles and the presence of drug encapsulation in an amorphous state. In vitro release studies showed that both formulations followed the same dissolution profiles, with no statistical differences. In cytotoxicity tests, IC50 values of 0.12 µM, 0.73 µM, and 4.06 µM were found for the formulation without TPGS, with TPGS, and pure drug, respectively, indicating the potentiation of the cytotoxic effect of resveratrol when encapsulated. Flow cytometry and confocal microscopy tests indicated excellent cellular uptake dependent on the concentration of nanoparticles, with a significant difference between the two formulations, suggesting that TPGS may pose a problem in the endocytosis of nanoparticles. The in vivo study evaluating the antitumor activity of the nanoparticles confirmed the data obtained in the in vitro tests, demonstrating that the nanoparticle without TPGS significantly reduced tumor volume, tumor mass, maintained body weight, and improved survival in mice. Moreover, the biochemical evaluation evidenced possible hepatotoxicity for formulation with TPGS.

Nanoencapsulation of Methylene-Blue for Enhanced Skin Cancer Cell Phototoxicity and Cutaneous Penetration in Association with Sonophoresis.

Photodynamic therapy (PDT) using methylene blue (MB) as a photosensitizer has emerged as an alternative treatment for skin cancers, such as squamous cell carcinoma (SCC). To increase the cutaneous penetration of the drug, some strategies are used, such as the association of nanocarriers and physical methods. Thus, herein we address the development of nanoparticles based on poly-Ɛ-caprolactone (PCL), optimized with the Box-Behnken factorial design, for topical application of MB associated with sonophoresis. The MB-nanoparticles were developed using the double emulsification-solvent evaporation technique and the optimized formulation resulted in an average size of 156.93 ± 8.27 nm, a polydispersion index of 0.11 ± 0.05, encapsulation efficiency of 94.22 ± 2.19% and zeta potential of -10.08 ± 1.12 mV. Morphological evaluation by scanning electron microscopy showed spherical nanoparticles. In vitro release studies show an initial burst compatible with the first-order mathematical model. The nanoparticle showed satisfactory generation of reactive oxygen species. The MTT assay was used to assess cytotoxicity and IC50; values of 79.84; 40.46; 22.37; 9.90 µM were obtained, respectively, for the MB-solution and the MB-nanoparticle without and with light irradiation after 2 h of incubation. Analysis using confocal microscopy showed high cellular uptake for the MB-nanoparticle. With regard to skin penetration, a higher concentration of MB was observed in the epidermis + dermis, corresponding to 9.81, 5.27 µg/cm2 in passive penetration and 24.31 and 23.81 µg/cm2 after sonophoresis, for solution-MB and nanoparticle-MB, respectively. To the best of our knowledge, this is the first report of MB encapsulation in PCL nanoparticles for application in skin cancer using PDT.

DOPE/CHEMS-Based EGFR-Targeted Immunoliposomes for Docetaxel Delivery: Formulation Development, Physicochemical Characterization and Biological Evaluation on Prostate Cancer Cells.

Docetaxel (DTX) is a non-selective antineoplastic agent with low solubility and a series of side effects. The technology of pH-sensitive and anti-epidermal growth factor receptor (anti-EGFR) immunoliposomes aims to increase the selective delivery of the drug in the acidic tumor environment to cells with EFGR overexpression. Thus, the study aimed to develop pH-sensitive liposomes based on DOPE (dioleoylphosphatidylethanolamine) and CHEMS (cholesteryl hemisuccinate), using a Box-Behnken factorial design. Furthermore, we aimed to conjugate the monoclonal antibody cetuximab onto liposomal surface, as well as to thoroughly characterize the nanosystems and evaluate them on prostate cancer cells. The liposomes prepared by hydration of the lipid film and optimized by the Box-Behnken factorial design showed a particle size of 107.2 ± 2.9 nm, a PDI of 0.213 ± 0.005, zeta potential of -21.9 ± 1.8 mV and an encapsulation efficiency of 88.65 ± 20.3%. Together, FTIR, DSC and DRX characterization demonstrated that the drug was properly encapsulated, with reduced drug crystallinity. Drug release was higher in acidic pH. The liposome conjugation with the anti-EGFR antibody cetuximab preserved the physicochemical characteristics and was successful. The liposome containing DTX reached an IC50 at a concentration of 65.74 nM in the PC3 cell line and 28.28 nM in the DU145 cell line. Immunoliposome, in turn, for PC3 cells reached an IC50 of 152.1 nM, and for the DU145 cell line, 12.60 nM, a considerable enhancement of cytotoxicity for the EGFR-positive cell line. Finally, the immunoliposome internalization was faster and greater than that of liposome in the DU145 cell line, with a higher EGFR overexpression. Thus, based on these results, it was possible to obtain a formulation with adequate characteristics of nanometric size, a high encapsulation of DTX and liposomes and particularly immunoliposomes containing DTX, which caused, as expected, a reduction in the viability of prostate cells, with high cellular internalization in EGFR overexpressing cells.

Nanoencapsulation of R-phycoerytrin extracted from Solieria filiformis improves protein stability and enables its biological application as a fluorescent dye.

We aimed to encapsulate R-PE to improve its stability for use as a fluorescent probe for cancer cells. Purified R-PE from the algae Solieria filiformis was encapsulated in polymeric nanoparticles using PCL. Nanoparticles were characterised and R-PE release was evaluated. Also, cellular uptake using breast and prostate cancer cells were performed. Nanoparticles presented nanometric particle size (198.8 ± 0.06 nm) with low polydispersity (0.13 ± 0.022), negative zeta potential (-18.7 ± 1.10 mV), and 50.0 ± 7.3% encapsulation. FTIR revealed that R-PE is molecularly dispersed in PCL. DSC peak at 307 °C indicates the presence of R-PE in the nanoparticle. Also, in vitro, it was demonstrated low release for nanoparticles and degradation for the free R-PE. Finally, cellular uptake demonstrated the potential of R-PE/PCL nanoparticles for cancer cell detection. Nanoparticles loaded with R-PE can overcome instability and allow application as a fluorescent probe for cancer cells.

Sorbitan Monolaurate-Containing Liposomes Enhance Skin Cancer Cell Cytotoxicity and in Association with Microneedling Increase the Skin Penetration of 5-Fluorouracil.

Squamous cell carcinoma (SCC) represents 20% of cases of non-melanoma skin cancer, and the most common treatment is the removal of the tumor, which can leave large scars. 5-Fluorouracil (5FU) is a drug used in the treatment of SCC, but it is highly hydrophilic, resulting in poor skin penetration in topical treatment. Some strategies can be used to increase the cutaneous penetration of the drug, such as the combination of liposomes containing penetration enhancers, for instance, surfactants, associated with the use of microneedling. Thus, the present work addresses the development of liposomes with penetration enhancers, such as sorbtitan monolaurate, span 20, for topical application of 5-FU and associated or not with the use of microneedling for skin delivery. Liposomes were developed using the lipid film hydration, resulting in particle size, polydispersity index, zeta potential, and 5-FU encapsulation efficiency of 88.08 nm, 0.169, -12.3 mV, and 50.20%, respectively. The presence of span 20 in liposomes potentiated the in vitro release of 5-FU. MTT assay was employed for cytotoxicity evaluation and the IC50 values were 0.62, 30.52, and 24.65 µM for liposomes with and without span 20 and 5-FU solution, respectively after 72-h treatment. Flow cytometry and confocal microscopy analysis evidenced high cell uptake for the formulations. In skin penetration studies, a higher concentration of 5-FU was observed in the epidermis + dermis, corresponding to 1997.71, 1842.20, and 2585.49 ng/cm2 in the passive penetration and 3214.07, 2342.84, and 5018.05 ng/cm2 after pretreatment with microneedles, for solution, liposome without and with span 20, respectively. Therefore, herein, we developed a nanoformulation for 5-FU delivery, with suitable physicochemical characteristics, potent skin cancer cytotoxicity, and cellular uptake. Span 20-based liposomes increased the skin penetration of 5-FU in association of microneedling. Altogether, the results shown herein evidenced the potential of the liposome containing span 20 for topical delivery of 5-FU.

Reversed-phase high-performance liquid chromatography: A fast and efficient analytical method to quantify docetaxel-loaded pegylated liposomes in release study.

Docetaxel is an anticancer that belongs to the family of taxanes and acts in the inhibition of cell proliferation through the polymerization of microtubules. The aim of this study was the development and validation of a fast method by reversed-phase high-performance liquid chromatography for quantitative analysis of docetaxel encapsulated in pegylated liposomes. The analytical method was validated for the following recognized specifications: system suitability, precision (repeatability and intermediate precision), linearity, accuracy, selectivity, detection and quantification limits, and robustness. The reversed phase-high-performance liquid chromatography analyses were performed at a temperature of 45°C (isocratic mode). The mobile phase was composed of acetonitrile and water (65:35, v/v) and the flow rate was fixed at 0.8 mL/min. The running time and wavelength were 8 min and 230 nm, respectively. The method was found to be linear, precise, selective, precise, robust, accurate, in the range of 1-75 µg/mL (R2 = 0.9999) and the values of detection and quantification limits were 2.35 and 7.84 µg/mL, respectively. The release rates of docetaxel in pegylated liposomes were lower compared to docetaxel in solution. The reversed phase high-performance liquid chromatography method developed proved to be adequate and can be effectively used to determine the in vitro release profile of docetaxel transported by pegylated liposomes.

A Review of Properties, Delivery Systems and Analytical Methods for the Characterization of Monomeric Glycoprotein Transferrin.

Transferrin is a protein involved in iron uptake by cells and has been identified as a potential target for directing drug-loaded nanoparticles for cancer treatment and diagnosis. Most methods for conjugation of transferrin and nanoparticles involve the formation of a thioeter bond between thiolated transferrin and maleimide-containing nanoparticle. For nanoparticle development, it is important to perform a thorough physicochemical characterization, including quantification of the amount of transferrin functionalizing the delivery system. Thus, following the transferrin and nanoparticle chemical conjugation, an analytical method is need for transferrin quantification. Altogether, we revised both physicochemical and pharmacokinetics transferrin characteristics, the aspects of iron transport after interaction with transferrin, the development of transferrin targeted-nanoparticles, highlighting both their composition, synthesis methods and in vitro/in vivo evaluation. Furthermore, we addressed the analytical methods employed in protein quantification, including spectrophotometric/colorimetric, immunoassays, electrophoretic and chromatographic techniques used to identify and/or quantify of transferrin in biological matrices and drug delivery systems.

Immunoconjugates for Cancer Targeting: A Review of Antibody-Drug Conjugates and Antibody-Functionalized Nanoparticles.

Targeted therapy has been recently highlighted due to the reduction of side effects and improvement in overall efficacy and survival from different types of cancers. Considering the approval of many monoclonal antibodies in the last twenty years, cancer treatment can be accomplished by the combination of monoclonal antibodies and small molecule chemotherapeutics. Thus, strategies to combine both drugs in a single administration system are relevant in the clinic. In this context, two strategies are possible and will be further discussed in this review: antibody-drug conjugates (ADCs) and antibody-functionalized nanoparticles. First, it is important to better understand the possible molecular targets for cancer therapy, addressing different antigens that can selectively bind to antibodies. After selecting the best target, ADCs can be prepared by attaching a cytotoxic drug to an antibody able to target a cancer cell antigen. Briefly, an ADC will be formed by a monoclonal antibody (MAb), a cytotoxic molecule (cytotoxin) and a chemical linker. Usually, surface-exposed lysine or the thiol group of cysteine residues are used as anchor sites for linker-drug molecules. Another strategy that should be considered is antibody-functionalized nanoparticles. Basically, liposomes, polymeric and inorganic nanoparticles can be attached to specific antibodies for targeted therapy. Different conjugation strategies can be used, but nanoparticles coupling between maleimide and thiolated antibodies or activation with the addition of ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/ N-hydroxysuccinimide (NHS) (1:5) and further addition of the antibody are some of the most used strategies. Herein, molecular targets and conjugation strategies will be presented and discussed to better understand the in vitro and in vivo applications presented. Also, the clinical development of ADCs and antibody-conjugated nanoparticles are addressed in the clinical development section. Finally, due to the innovation related to the targeted therapy, it is convenient to analyze the impact on patenting and technology. Information related to the temporal evolution of the number of patents, distribution of patent holders and also the number of patents related to cancer types are presented and discussed. Thus, our aim is to provide an overview of the recent developments in immunoconjugates for cancer targeting and highlight the most important aspects for clinical relevance and innovation.

EGFR targeting for cancer therapy: Pharmacology and immunoconjugates with drugs and nanoparticles.

The epidermal growth factor receptor (EGFR) belongs to the tyrosine kinase receptors family and is present in the epithelial cell membrane. Its endogenous activation occurs through the binding of different endogenous ligands, including the epidermal growth factor (EGF), leading to signaling cascades able to maintain normal cellular functions. Although involved in the development and maintenance of tissues in normal conditions, when EGFR is overexpressed, it stimulates the growth and progression of tumors, resulting in angiogenesis, invasion and metastasis, through some main cascades such as Ras/Raf/MAPK, PIK-3/AKT, PLC-PKC and STAT. Besides, considering the limitations of conventional chemotherapy that result in high toxicity and low tumor specificity, EGFR is currently considered an important target. As a result, several monoclonal antibodies are currently approved for use in cancer treatment, such as cetuximab (CTX), panitumumab, nimotuzumab, necitumumab and others are in clinical trials. Aiming to combine the chemotherapeutic agent toxicity and specific targeting to EGFR overexpressing tumor tissues, two main strategies will be discussed in this review: antibody-drug conjugates (ADCs) and antibody-nanoparticle conjugates (ANCs). Briefly, ADCs consist of antibodies covalently linked through a spacer to the cytotoxic drug. Upon administration, binding to EGFR and endocytosis, ADCs suffer chemical and enzymatic reactions leading to the release and accumulation of the drug. Instead, ANCs consist of nanotechnology-based formulations, such as lipid, polymeric and inorganic nanoparticles able to protect the drug against inactivation, allowing controlled release and also passive accumulation in tumor tissues by the enhanced permeability and retention effect (EPR). Furthermore, ANCs undergo active targeting through EGFR receptor-mediated endocytosis, leading to the formation of lysosomes and drug release into the cytosol. Herein, we will present and discuss some important aspects regarding EGFR structure, its role on internal signaling pathways and downregulation aspects. Then, considering that EGFR is a potential therapeutic target for cancer therapy, the monoclonal antibodies able to target this receptor will be presented and discussed. Finally, ADCs and ANCs state of the art will be reviewed and recent studies and clinical progresses will be highlighted. To the best of our knowledge, this is the first review paper to address specifically the EGFR target and its application on ADCs and ANCs.

Poly-ε-caprolactone Nanoparticles Loaded with 4-Nerolidylcatechol (4-NC) for Growth Inhibition of Microsporum canis.

Dermatophyte fungal infections are difficult to treat because they need long-term treatments. 4-Nerolidylcatechol (4-NC) is a compound found in Piper umbellatum that has been reported to demonstrate significant antifungal activity, but is easily oxidizable. Due to this characteristic, the incorporation in nanostructured systems represents a strategy to guarantee the compound's stability compared to the isolated form and the possibility of improving antifungal activity. The objective of this study was to incorporate 4-NC into polymeric nanoparticles to evaluate, in vitro and in vivo, the growth inhibition of Microsporum canis. 4-NC was isolated from fresh leaves of P. umbellatum, and polymer nanoparticles of polycaprolactone were developed by nanoprecipitation using a 1:5 weight ratio (drug:polymer). Nanoparticles exhibited excellent encapsulation efficiency, and the antifungal activity was observed in nanoparticles with 4-NC incorporated. Polymeric nanoparticles can be a strategy employed for decreased cytotoxicity, increasing the stability and solubility of substances, as well as improving the efficacy of 4-NC.

Nanoencapsulation of triterpene 3ß,6ß,16ß-trihydroxylup-20(29)-ene from Combretum leprosum as strategy to improve its cytotoxicity against cancer cell lines.

The pentacyclic triterpene 3ß,6ß,16ß-tri-hydroxilup-20(29)-ene is a natural product produced by the Brazilian medicinal plant Combretum leprosum. Its cytotoxicity has been previously reported against breast cancer cell lines. The low water solubility of this natural product, that hampers its bioavailability, motivated the investigation of a new nanoparticle formulation containing the triterpene in order to improve its bioactivity. The triterpene was encapsulated in polycaprolactone (PCL) polymer by nanoprecipitation, producing homogenic nanoparticles with nanometer sizes (122.7 ± 2.06 nm), which were characterized by FT-IR, SEM imaging and DSC. The cytotoxicity (MTT method) of the nanoparticle containing the triterpene 1, besides the free natural product and the nanoparticle control (without 1), was assayed against three human tumor cell lines [human colon carcinoma line (HCT116), prostate (PC3) and glioblastoma (SNB19)] and the normal epithelial embryo kidney human cell line (Hek293T). The nanocarrier produced a significative effect in the cytotoxicity of the natural product in the nanoformulation (IC50 0.11-0.26 µg mL-1) when compared with its free form (IC50 1.07-1.44 µg mL-1). Additionally, higher selectivity of the triterpene to the tumor cells was found when it was encapsulated (SI 1.92-4.54) than in its free form (SI 0.42-0.56). In this case, the nanoencapsulated triterpene was more selective to PC3 (SI 3.33) and SNB19 (SI 4.54) tumor cells.

EGFR-targeted immunoliposomes efficiently deliver docetaxel to prostate cancer cells.

Prostate cancer is the second cause of cancer death in men worldwide. Docetaxel (DTX), an antimitotic drug, is widely used for the treatment of metastatic prostate cancer patients. Taxotere® is a commercial DTX formulation. It contains a polysorbate 80 surfactant to improve DTX aqueous solubility, which has been associated with hypersensitivity reactions in patients. Liposomes have been used as promising delivery systems for a range of hydrophobic drugs, such as DTX, offering improved drug water solubility and biocompatibility, without compromising its anticancer activity. Herein, DTX-loaded liposomes were developed using the Box-Behnken factorial design. The optimized formulation was nano-sized, homogenous in size (67.47 nm) with high DTX encapsulation efficiency (99.95 %). The encapsulated DTX was in a soluble amorphous state, which was slowly released. Next, to increase the liposomes selectivity to prostate cancer cells, cetuximab, an anti-EGFR monoclonal antibody. was successfully conjugated to the surface of liposomes, without compromising cetuximab protein structure and stability. As expected, our results showed higher cellular uptake and toxicity of immunoliposomes, compared to non-targeted liposomes, in DU145 (EGFR-overxpressing) prostate cancer cells. To the best of our knowledge, this is the first report of engineering EGFR-targeted liposomes to enhance the selectivity of DTX delivery to EGFR-positive prostate cancer cells.

Synthesis and Characterization of Nanostructured Lipid Nanocarriers for Enhanced Sun Protection Factor of Octyl p-methoxycinnamate.

Sunlight is important to health, but higher exposure to radiation causes early aging of the skin and skin damage that can lead to skin cancers. This study aimed at producing a stable octyl p-methoxycinnamate (OMC)-loaded nanostructured lipid carrier (NLC) sunscreen, which can help in the photoprotective effect. NLC was produced by emulsification-sonication method and these systems were composed of myristyl myristate (MM), caprylic capric triglyceride (CCT), Tween® 80 (TW), and soybean phosphatidylcholine (SP) and characterized by dynamic light scattering (DLS), zeta potential (ZP) measurement, atomic force microscopy (AFM), scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and in vitro release studies. Pre-formulation studies were performed changing TW concentrations and no differences were found at concentrations of 1.0 and 2.0%. Two selected formulations were designed and showed an average size of 91.5-131.7, polydispersity index > 0.2, and a negative value of ZP. AFM presented a sphere-like morphology and SEM showed ability to form a thin film. DSC exhibited that the incorporation of OMC promoted reduction of enthalpy due to formation of a more amorphous structure. Drug release shows up to 55.74% and 30.57%, and this difference could be related to the presence of SP in this formulation that promoted a more amorphous structure; the release mechanism study indicated Fickian diffusion and relaxation. Sun protection factor (SPF) evaluation was performed using NLC and presented values around 40, considerably higher than those observed in the literature. The developed formulations provide a beneficial alternative to conventional sunscreen formulations.

Stimuli-responsive Drug Delivery Nanocarriers in the Treatment of Breast Cancer.

Stimuli-responsive drug-delivery nanocarriers (DDNs) have been increasingly reported in the literature as an alternative for breast cancer therapy. Stimuli-responsive DDNs are developed with materials that present a drastic change in response to intrinsic/chemical stimuli (pH, redox and enzyme) and extrinsic/physical stimuli (ultrasound, Near-infrared (NIR) light, magnetic field and electric current). In addition, they can be developed using different strategies, such as functionalization with signaling molecules, leading to several advantages, such as (a) improved pharmaceutical properties of liposoluble drugs, (b) selectivity with the tumor tissue decreasing systemic toxic effects, (c) controlled release upon different stimuli, which are all fundamental to improving the therapeutic effectiveness of breast cancer treatment. Therefore, this review summarizes the use of stimuli-responsive DDNs in the treatment of breast cancer. We have divided the discussions into intrinsic and extrinsic stimuli and have separately detailed them regarding their definitions and applications. Finally, we aim to address the ability of these stimuli-responsive DDNs to control the drug release in vitro and the influence on breast cancer therapy, evaluated in vivo in breast cancer models.

A Critical Review of Properties and Analytical/Bioanalytical Methods for Characterization of Cetuximab.

Cetuximab (CTX) is a chimeric monoclonal antibody (mAb) able to selectively bind to the epidermal growth factor receptor (EGFR), resulting in inhibition of EGF linkage and phosphorylation cascade interruption. As a result, it is able to prevent cell proliferation, angiogenesis and metastasis, usually related to cancer malignization. As the EGFR is overexpressed in many human tumors, its use has been approved by FDA since 2006. Clinical use of CTX has been proved to cause skin rash which is related to the better prognosis. Thus, currently strategies also focus on the development of safe and effective drug delivery systems and on quantification methods for CTX in a variety of matrices. Based on the challenges to quantify CTX, immunoassays, spectrophotometric assays, electrophoretic assays and chromatographic assays are under study. Among them, the spectrophotometric/colorimetric techniques, used in near 32% of the papers investigated, followed by chromatographic techniques and immunoassay methods, such as enzyme-linked immunosorbent assay (ELISA), used in 29% and 26%, respectively, and electrophoretic techniques used in near 13%. Herein, we will describe and discuss CTX main aspects and highlight the main quantification methods that are currently used for its quantification in different matrices.

Poly-ε-caprolactone Nanoparticles Loaded with 4-Nerolidylcatechol (4-NC) for Growth Inhibition of Microsporum canis

Dermatophyte fungal infections are difficult to treat because they need long-term treatments. 4-Nerolidylcatechol (4-NC) is a compound found in Piper umbellatum that has been reported to demonstrate significant antifungal activity, but is easily oxidizable. Due to this characteristic, the incorporation in nanostructured systems represents a strategy to guarantee the compound's stability compared to the isolated form and the possibility of improving antifungal activity. The objective of this study was to incorporate 4-NC into polymeric nanoparticles to evaluate, in vitro and in vivo, the growth inhibition of Microsporum canis. 4-NC was isolated from fresh leaves of P. Umbellatum, and polymer nanoparticles of polycaprolactone were developed by nanoprecipitation using a 1:5 weight ratio (drug:polymer). Nanoparticles exhibited excellent encapsulation efficiency, and the antifungal activity was observed in nanoparticles with 4-NC incorporated. Polymeric nanoparticles can be a strategy employed for decreased cytotoxicity, increasing the stability and solubility of substances, as well as improving the efficacy of 4-NC(AU).

Targeted Liposomes for siRNA Delivery to Cancer.

BACKGROUND: RNA interference is a promising therapeutic tool for the treatment of a variety of diseases, with great potential for cancer therapy. Small interfering RNA (siRNA), however, presents several drawbacks that hamper its therapeutic application. Lipid nanoparticles, including liposomes, are delivery systems with great potential for siRNA delivery, protecting it from degradation, enhancing its cell uptake with the ability of controlled release. However, non-specific delivery and side effects could potentially limit the in vivo application. Therefore, targeting lipid nanoparticles to overexpressed receptors of cancer cells represents a strategy for better therapeutic outcome, with improved efficacy and reduced toxicity. For this purpose, lipid nanoparticles could be functionalized with several moieties that can be recognized by cancer cells more than by normal cells. These ligands include folate, transferrin, peptides, oligosaccharides, monoclonal antibodies and aptamers. METHODS: In this paper, we reviewed functionalization strategies and addressed the major in vitro and in vivo findings in the field of cancer treatment with siRNA. RESULTS: Many papers showed enhanced siRNA delivery by targeted liposomes, resulting in enhanced drug uptake and better cytotoxicity, with consequent better tumor growth control in xenograft studies. CONCLUSION: siRNA delivery mediated by functionalized liposomes is promising, but clinical trials need to be conducted.

Skin cancer treatment effectiveness is improved by iontophoresis of EGFR-targeted liposomes containing 5-FU compared with subcutaneous injection.

Squamous cell carcinoma (SCC) is a malignant tumor in which epidermal growth factor receptor (EGFR) overexpression is associated with poor prognosis and malignancy. For SCC treatment, cetuximab, an anti-EGFR antibody, is administered in combination with a chemotherapeutic drug for improved efficacy. In this work, an EGFR-targeted immunoliposome loaded with 5-fluorouracil (5- FU) was developed to allow co-administration of the antibody and the chemotherapeutic agent and selective delivery to SCC cells. Topically applied iontophoresis and subcutaneous injections of the 5-FU-loaded immunoliposomes were employed in an SCC xenograft animal model to evaluate the influence of the administration route on therapeutic efficacy. In vitro, cellular uptake of cetuximab-immunoliposomes by EGFR-positive SCC cells was 3.5-fold greater than the uptake of control liposomes. Skin penetration studies showed that iontophoresis of immunoliposomes doubled the 5-FU penetration into the viable epidermis compared with the same treatment with control liposomes. In vivo, subcutaneous injection of immunoliposomes reduced tumor volume by >60% compared with the negative control and approximately 50% compared with the 5-FU solution and control liposome treatments. Interestingly, topical administration via iontophoresis improved tumor reduction by almost 2-fold compared with subcutaneous administration of 5-FU solution and control liposomes but was equally effective for the immunoliposome treatment. However, histological analysis showed that iontophoresis of immunoliposomes was more effective than subcutaneous injection in reducing cell proliferation, resulting in cells with less aggressive characteristics. In conclusion, topical administration of immunoliposomes containing 5-FU using iontophoresis is a promising strategy for SCC treatment.

A Critical Review of Properties and Analytical Methods for the Determination of Docetaxel in Biological and Pharmaceutical Matrices.

Docetaxel (DTX) is an antineoplastic agent of the second generation of the taxoid family. It is a semi-synthetic drug prepared from a precursor extracted of the plant Taxus baccata. The commercial formulation of DTX, Taxotere®, employs the surfactant polysorbate 80, due to the low water solubility of the drug, causing several side effects. Therefore, there is a need to develop delivery systems to reduce the side effects of DTX. In addition, this drug has been qualitative and quantitatively analyzed in pharmaceutical formulations and biological samples. Thus, several techniques and analytical methods have been reported with the aim of optimizing the analytical signal, increasing sensitivity, selectivity and reducing the effects of interference. Herein, we highlight immunoassay, capillary electrophoresis and chromatographic methods. This review presents a summary of physicochemical and pharmacokinetics properties, mechanisms of action, drug delivery systems and analytical methods used in quantification of DTX in diverse matrices such as blood, plasma, oral fluid, urine, carcinoma cells, pharmaceutical formulations and delivery systems.