Application of Asymmetrical Flow Field-Flow Fractionation for Characterizing the Size and Drug Release Kinetics of Theranostic Lipid Nanovesicles

Liposome size and in vitro release of the active substance belong to critical quality attributes of liposomal carriers. Here, we apply asymmetric flow field-flow fractionation (AF4) to characterize theranostic liposomes prepared by thin lipid film hydration/extrusion or microfluidics. The vesicles’ size was derived from multi-angle laser light scattering following fractionation (AF4) and compared to sizes derived from dynamic light scattering measurements. Additionally, we adapted a previously developed AF4 method to study zinc phthalocyanine (ZnPc) release/transfer from theranostic liposomes. To this end, theranostic liposomes were incubated with large acceptor liposomes serving as a sink (mimicking biological sinks) and were subsequently separated by AF4. During incubation, ZnPc was transferred from donor to acceptor fraction until reaching equilibrium. The process followed first-order kinetics with half-lives between 119.5–277.3 min, depending on the formulation. The release mechanism was postulated to represent a combination of Fickian diffusion and liposome relaxation. The rate constant of the transfer was proportional to the liposome size and inversely proportional to the ZnPc/POPC molar ratio. Our results confirm the usefulness of AF4 based method to study in vitro release/transfer of lipophilic payload, which may be useful to estimate the unwanted loss of drug from the liposomal carrier in vivo

Development of Liposomal Vesicles for Osimertinib Delivery to EGFR Mutation—Positive Lung Cancer Cells

Osimertinib (OSI, AZD9291), is a third-generation, irreversible tyrosine kinase inhibitor (TKI) of the epidermal growth factor receptor (EGFR) that selectively inhibits both EGFR-TKI–sensitizing and EGFR T790M resistance mutations. OSI has been approved as a first-line treatment of EGFR-mutant lung cancer and for metastatic EGFR T790M-mutant non-small cell lung cancer. Liposome-based delivery of OSI can provide a new formulation of the drug that can be administered via alternative delivery routes (intravenous, inhalation). In this manuscript, we report for the first time development and characterization of liposomal OSI formulations with diameters of ca. 115 nm. Vesicles were composed of phosphatidylcholines with various saturation and carbon chain lengths, cholesterol and pegylated phosphoethanolamine. Liposomes were loaded with OSI passively, resulting in a drug being dissolved in the phospholipid matrix or actively via remote-loading leading to the formation of OSI precipitate in the liposomal core. Remotely loaded liposomes were characterized by nearly 100% entrapment efficacy and represent a depot of OSI. Passively-loaded vesicles released OSI following the Peppas-Sahlin model, in a mechanism combining drug diffusion and liposome relaxation. OSI-loaded liposomes composed of l-α-phosphatidylcholine (egg-PC) demonstrated a higher toxicity in non-small lung cancer cells with EGFR T790M resistance mutation (H-1975) when compared with free OSI. Developed OSI formulations did not show antiproliferative activity in vitro in healthy lung epithelial cells (MRC-5) without the EGFR mutation

Liposomal Zn- and Al-phthalocyanine enhance photodynamic therapy of oral cancer

Objective: Photodynamic therapy (PDT) has been studied as a promising method to eliminate cancer cells. We used liposomes as a method of specific drug delivery for PDT and studied the effectiveness of the liposome-encapsulated photosensitizers, zinc phthalocyanine (ZnPc) and aluminum phthalocyanine chloride (AlPc), on oral squamous cell carcinoma. Methods: Liposomes were composed of palmitoyloleoyphosphatidylcholine (POPC): phosphatidylglycerol (PG), and contained either ZnPc or AlPc. Free or liposome-encapsulated ZnPc and AlPC were added to HSC-3 cells in the concentration range 0.1-1 µM, and incubated for 24 h at 37°C. The cells were then exposed to light (690 nm) from a High Power LED Multi Chip Emitter. Cytotoxicity was evaluated by the Alamar Blue assay that measures metabolic activity, using a Molecular Devices Versamax microplate reader. Results: Cells treated with ZnPc and AlPc encapsulated in liposomes resulted in further decrease in cell viability when compared to cells treated with free ZnPc and AlPc. The Alamar Blue assay showed a linear reduction with increased concentrations of ZnPc and AlPc in both free and liposomal form. For 0.1, 0.5, and 1 µM liposomal ZnPc, the viability was reduced to 89±4, 69±4, and 41±5%, respectively. With free ZnPc, the values were 104±5, 89±5, and 75±6%, respectively. For 0.1, 0.5, 1 µM liposomal AlPc, the viability was reduced to 54±3, 20±3, and 21±2%, respectively. With free AlPc, the viabilities were 108±5, 78±8, and 51±1%, respectively. Conclusion: HSC-3 cells are vulnerable to liposomal ZnPc and AlPc in a dose-dependent manner, following light activation. Liposomal ZnPc and AlPc both reduce cell metabolic activity more effectively than the free photosensitizers. Our studies indicate that liposomal delivery of ZnPc and AlPc results in a more efficient elimination of oral squamous cell carcinoma

Photodynamic therapy of Porphyromonas gingivalis via liposome-encapsulated sensitizers

Photodynamic therapy exploits the light-activation of a photosensitizer to cause cytotoxicity. Liposomes can be used to deliver hydrophobic photosensitizers to bacteria. Positively charged dioleoyltrimethylammoniumpropane:palmitoyloleoylphosphatidylcholine (1:1) liposomes bound quantitatively to the periodontal pathogen, Porphyromonas gingivalis. Following illumination, free and liposomal zinc phthalocyanine reduced the colony-forming unit (CFU) to 65 percent and 23 percent of controls, respectively. Thus, localization of the photosensitizer at the surface of bacteria via liposome binding enhanced the photodynamic cytotoxicity of zinc phthalocyanine

Photodynamic therapy of Porphyromonas gingivalis via liposome-encapsulated sensitizers

Photodynamic therapy exploits the light-activation of a photosensitizer to cause cytotoxicity. Liposomes can be used to deliver hydrophobic photosensitizers to bacteria. Positively charged dioleoyltrimethylammoniumpropane:palmitoyloleoylphosphatidylcholine (1:1) liposomes bound quantitatively to the periodontal pathogen, Porphyromonas gingivalis. Following illumination, free and liposomal zinc phthalocyanine reduced the colony-forming unit (CFU) to 65 percent and 23 percent of controls, respectively. Thus, localization of the photosensitizer at the surface of bacteria via liposome binding enhanced the photodynamic cytotoxicity of zinc phthalocyanine

Photosensitizers mediated photodynamic inactivation against virus particles

Viruses cause many diseases in humans from the rather innocent common cold to more serious or chronic, life-threatening infections. The long-term sideeffects, sometimes low effectiveness of standard pharmacotherapy and the emergence of drug resistance require a search for new alternative or complementary antiviral therapeutic approaches. One new approach to inactivate microorganisms is photodynamic antimicrobial chemotherapy (PACT). PACT has evolved as a potential method to inactivate viruses. The great challenge for PACT is to develop a methodology enabling the effective inactivation of viruses while leaving the host cells as untouched as possible. This review aims to provide some main directions of antiviral PACT, taking into account different photosensitizers, which have been widely investigated as potential antiviral agents. In addition, several aspects concerning PACT as a tool to assure viral inactivation in human blood products will be addressed.status: publishe

Photodynamic Therapy of Oral Cancer and Novel Liposomal Photosensitizers

Photodynamic therapy facilitates the selective destruction of cancer tissue by utilizing a photosensitizer drug, the light near the absorbance wavelength of the drug, and oxygen. Methylene Blue, 5-aminolevulinic acid (the precursor of the photosensitizer, protoporphyrin IX), porphyrin, Foscan, Chlorin e6, and HPPH have been used successfully as photosensitizers in the treatment of oral verrucous hyperplasia, oral leukoplakia, oral lichen planus, and head and neck squamous cell carcinoma. “Theranostic” liposomes can deliver a contrast agent for magnetic resonance imaging and a photosensitizer for the image-guided photodynamic therapy of head and neck cancer. Liposomes incorporating photosensitizers can be targeted to cell surface markers overexpressed on cancer cells. Novel porphyrinoids have been developed in our laboratories that are highly effective as photosensitizers. Tribenzoporphyrazines encapsulated in cationic liposomes have produced IC50 values up to 50 times lower compared to the free photosensitizers. It is anticipated that targeting these drugs to cancer stem cells, using upconversion nanoparticles for the near-infrared irradiation of tumors to activate the photosensitizers, and overcoming tumor hypoxia will enhance the efficacy of photodynamic therapy of tumors accessible to light sources

Cellular changes, molecular pathways and the immune system following photodynamic treatment

Photodynamic therapy (PDT) is a novel medical technique involving three key components: light, a photosensitizer molecule and molecular oxygen, which are essential to achieve the therapeutic effect. There has been great interest in the use of PDT in the treatment of many cancers and skin disorders. Upon irradiation with light of a specific wavelength, the photosensitizer undergoes several reactions resulting in the production of reactive oxygen species (ROS). ROS may react with different biomolecules, causing defects in many cellular structures and biochemical pathways. PDT-mediated tumor destruction in vivo involves cellular mechanisms with photodamage of mitochondria, lysosomes, nuclei, and cell membranes that activate apoptotic, necrotic and autophagic signals, leading to cell death. PDT is capable of changing the tumor microenvironment, thereby diminishing the supply of oxygen, which explains the antiangiogenic effect of PDT. Finally, inflammatory and immune responses play a crucial role in the long-lasting consequences of PDT treatment. This review is focused on the biochemical effects exerted by photodynamic treatment on cell death signaling pathways, destruction of the vasculature, and the activation of the immune system

Photodynamic therapy of cancer with liposomal photosensitizers

The photodynamic reaction involves the light-induced generation of an excited state in a photosensitizer molecule (PS), which then results in the formation of reactive oxygen species in the presence of oxygen, or a direct modification of a cellular molecule. Most PSs are porphyrinoids, which are highly lipophilic, and are administered usually in liposomes to facilitate their effective delivery to target cells. The currently available liposomal formulations are Visudyne® and Fospeg®. Novel PSs were developed and tested for their photodynamic activity against cancer cells. Several compounds were highly phototoxic to oral cancer cells both in free and liposome-encapsulated form, with nanomolar IC50 values. The lowest IC50s (7-13 nM) were obtained with a PS encapsulated in cationic liposomes