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.

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.

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.