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.

Liquid crystalline nanogel targets skin cancer via low-frequency ultrasound treatment.

The potential of low-frequency ultrasound (LFU) combined with nanotechnology-based formulations in improving skin tumors topical treatment was investigated. The impact of solid lipid nanoparticles (SLN) and hydrophilic nanogels as coupling media on LFU-induced skin localized transport regions (LTR) and the penetration of doxorubicin (DOX) in LFU-pretreated skin was evaluated. SLN were prepared by the microemulsion technique and liquid crystalline nanogels using Poloxamer. In vitro, the skin was pretreated with LFU until skin resistivity of âˆ¼1 KΩ.cm2 using the various coupling media followed by evaluation of DOX penetration from DOX-nanogel and SLN-DOX in skin layers. Squamous cell carcinoma (SCC) induced in mice was LFU-treated using the nanogel with the LFU tip placed 5 mm or 10 mm from the tumor surface, followed by DOX-nanogel application. LFU with nanogel coupling achieved larger LTR areas than LFU with SLN coupling. In LFU-pretreated skin, DOX-nanogel significantly improved drug penetration to the viable epidermis, while SLN-DOX hindered drug transport through LTR. In vivo, LFU-nanogel pretreatment with the 10 mm tip distance induced significant tumor inhibition and reduced tumor cell numbers and necrosis. These findings suggest the importance of optimizing nanoparticle-based formulations and LFU parameters for the clinical application of LFU technology in skin tumor treatment.

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.

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.

Production and characterization of alginate bilayer membranes for releasing simvastatin to treat wounds.

This study aims to produce and characterize alginate bilayer membranes composed of single membranes with varying cross-linking degrees to modulate simvastatin release, with potential to be used for wound-dressing. The single-layer and bilayer membranes were characterized by weight, thickness, surface pH, equilibrium-humidity, swelling degree, solubility, infrared spectroscopy (attenuated total reflectance Fourier-transform infrared), scanning electron microscopy, and water vapor transmission. Simvastatin diffusion and release rates were analyzed using Franz's cells; its indirect cytotoxicity was analyzed using human keratinocyte cells. The difference in the cross-linking degree (bottom and top layers) influenced the morphology of the membrane, and consequently its physical barrier properties. An in vitro release study demonstrated that the bilayer membrane could sustain drug-release for longer time as compared to the single-layer membrane, which could be potentially beneficial for long-term treatment of chronic wounds. A cell viability assay showed that simvastatin-loaded alginate membranes could be characterized as noncytotoxic, demonstrating their potential for use in wound-dressing applications.

The prominence of the dosage form design to treat ocular diseases.

The eye is susceptible to various diseases commonly difficult to treat. To overcome the barriers imposed by this organ for required drugs penetration, technological strategies have been implemented to ocular formulations. Among them are the use of temperature or electric stimuli and the development of nanoparticles. The objective of this review is to present the main barriers to ocular drug delivery and to discuss strategies used in the development of ocular dosage forms, primarily for topical delivery, to increase the local bioavailability of drugs, target their delivery and increase patient compliance. Results obtained in the last years related to the topical administration of liposomes, dendrimers, iontophoresis, among other nanoparticulate systems focused on ophthalmic delivery, will be addressed. Finally, some clinical trials and marketed formulations that use nanotechnology to topically treat eye diseases will be presented.

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.

Liposome-based nanocarrier loaded with a new quinoxaline derivative for the treatment of cutaneous leishmaniasis.

The use of nanocarriers for drug delivery is a strategy aimed to improve therapeutic indices through changes in their pharmacokinetic and pharmacodynamic characteristics. Liposomes are well-investigated nanocarriers for drug delivery to macrophage-targeted therapy, the main hosts of intracellular pathogens of some infectious diseases, such as leishmaniasis. In this study, we developed hyaluronic acid (HA)-coated liposomes by different methods that can encapsulate a new quinoxaline derivative, the LSPN331, to increase its solubility and improve its bioavailability. The surface modification of liposomes and their physicochemical characteristics may depend on the coating method, which may be a critical parameter with regard to the route of administration of the antileishmanial drug. Liposomes with identical phospholipid composition containing the same drug were developed, and different biological responses were verified, and our hypothesis is that it is related to the type of modification of the surface. Different physicochemical characterization techniques (dynamic light scattering, transmission electron microscopy and UV-vis quantification of labeled-HA) were used to confirm the successful modification of liposomes as well as their stability upon storage. The encapsulation of LSPN331 was performed using HPLC method, and the entrapment efficiency (EE%) was satisfatory in all formulations, considering results of similar formulations in the literature. Furthermore, in vitro and in vivo studies were carried out to evaluate the efficacy against the parasite Leishmania amazonensis. The in vitro activity was maintained or even improved and HA-coated liposomes showed the ability to target to the site of action by the proposed routes of administration, topically and intravenously. Both formulations are promising for future tests of antileishmania activity in vivo.

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.

Prospective insulin-based ophthalmic delivery systems for the treatment of dry eye syndrome and corneal injuries.

The presence of insulin (INS) receptors on the ocular surface (OS) and lacrimal gland (LG), and the high prevalence of dry eye syndrome (DES) and corneal lesions in diabetic patients suggest that INS is relevant for OS homeostasis and wound healing. The study aims at developing delivery systems for the topical administration of INS to the OS in order to improve INS local bioavailability and evaluate the influence of the delivery systems on DES in diabetic rats (DM) (n = 05/group). Chitosan microparticles (MP), chitosan/poloxamer gel (GEL) and MP-loaded GEL (GELMP), with or without INS were developed. Formulations were instilled into the eyes of diabetic rats (DM) for 15 days and the tear fluid volume, corneal cells morphology and cornea thickness were assessed and compared with an aqueous dispersion of INS (DISP-INS). All delivery systems had pH of about 6, osmolality suitable for topical application and positive zeta potential. The MPs with or without INS had sizes close to 4 µm, spherical morphology and INS encapsulation efficiency of 77 ±â€¯6%. DISP-INS and GELMP-INS formulations produced tear secretion amounts significantly higher than those receiving formulations containing no INS and similar to healthy animals. Cornea surface impression cytology showed that treatment with INS-delivery systems and not DISP-INS almost normalized cells morphology. Treatment with GELMP-INS increased INS by 2.5 in the LG and eyeball as compared to the groups treated with GEL-INS and MP-INS, while treatment with DISP-INS left no traces of drug in the eye after treatment termination. GEL and GELMP containing INS were also able to normalize the thickness of the corneal epithelia. In conclusion, GELMP-INS normalized tear fluid volume, corneal thickness, protected corneal cells morphology and increased ocular bioavailability of INS, making it a promising treatment strategy for DES and corneal lesions.

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.

Preparation of Immunoliposomes by Direct Coupling of Antibodies Based on a Thioether Bond.

Drug delivery is of paramount importance, since the drug needs to be delivered to a specific site, in adequate concentration, avoiding degradation in order to provide therapeutic efficacy. Different nanocarriers have been used over the years for this purpose and liposomes are well-established systems due to the high biocompatibility and the possibility to vehiculate both hydrophilic and lipophilic drugs. In order to circumvent the rapid clearance by the reticuloendothelial system and to avoid the healthy cells exposure to the drug, long circulating liposomes containing polyethyleneglycol (PEG) and functionalized liposomes for targeted delivery have been developed. Immunoliposomes consist of liposomes containing antibodies or antibody fragments attached at the membrane surface. This attachment can be performed using PEG lipids, containing a reactive terminal group such as maleimide and thiolated antibodies. Additionaly, the use of PEG chains as spacers increases antibody-antigen affinity, since the antibody is not shielded by the steric hindrance of PEG and also due to the correct orientation of antibodies for interaction with receptors on cell surface. In this chapter, we describe and discuss in details the protocol to prepare anti-epidermal growth factor receptor (anti-EGFR) and anti-human epidermal growth factor receptor 2 (anti-HER2) liposomes using cetuximab and trastuzumab as antibodies. We present the direct coupling method based on the maleimide thioether reaction for these immunoliposomes preparation and present some characterization steps and in vitro studies in cell culture which can be used for better understanding these nanocarriers.

Evaluation of critical parameters for in vitro skin permeation and penetration studies using animal skin models.

In vitro skin permeation/penetration studies may be affected by many sources of variation. Herein, we aimed to investigate the major critical procedures of in vitro skin delivery studies. These experiments were performed with model drugs according to official guidelines. The influence of skin source on penetration studies was studied as well as the use of a cryopreservation agent on skin freezing evaluated by transepidermal water loss, electrical resistance, permeation/penetration profiles and histological changes of the skin. The best condition for tape stripping procedure was validated through the evaluation of the distribution of corneocytes, mass of stratum corneum (SC) removed and amount of protein removed using finger pressure, a 2kg weight and a roller. The interchangeability of the tape stripping procedures followed by the epidermis and dermis homogenate and the micrometric horizontal cryostat skin sectioning methods were also investigated, besides the effect of different formulations. Noteworthy, different skin sources were able to ensure reliable interchangeability for in vitro permeation studies. Furthermore, an increased penetration was obtained for stored frozen skin compared to fresh skin, even with the addition of a cryoprotectant agent. The best method for tape stripping was the finger pressure followed by the addition of a propylene glycol solvent leading to better SC removal. Finally, no significant difference was found in skin penetration studies performed by different methods suggesting their possible interchangeability.

Physical methods for topical skin drug delivery: concepts and applications

Topical drug delivery is an interesting approach to treat skin diseases and to avoid pain and low patient compliance in cases where a systemic delivery is required. However, the stratum corneum, which is the outermost skin layer, strongly protects the body from the entrance of substances, especially those hydrophilic. In this context, different physical methods have been studied to overcome the stratum corneum barrier and facilitate penetration of drugs into or through the skin. Among them, iontophoresis, low-frequency ultrasound and microneedles have been widely employed for transdermal drug delivery. More recently, they are also studied to aid in the treatment of dermatological disorders, such as skin tumors and inflammation. Basically, iontophoresis refers to the movement of charged and non-charged hydrophilic molecules through the skin due to the application of a low constant electric current and the contributions of electromigration and electroosmosis. In low-frequency ultrasound, cavitation is the main mechanism for skin permeabilization that consists on the formation of microbubbles that disorganize the stratum corneum. Microneedles are microprojections, minimally invasive, that can be designed with different lengths, materials and geometry to increase skin permeability. In this review, concepts, mechanisms and applications of these three physical methods will be presented and discussed with focus on their use in dermatological treatments. Moreover, comparative studies using different physical methods will be presented and also some clinical perspectives will be addressed