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.

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.

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.

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.