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In the current study, it was demonstrated that three commercially available grades of hydrogenated phospholipids (HPL) differing in their content of phosphatidylcholine may be used as components for hot melt-extruded binary (HPL as sole excipient) or ternary (in combination with copovidone) solid dispersions of fenofibrate (FEN) at mass fractions between 0.5 and 20% (ternary) or 80% (binary). X-ray powder diffraction indicated complete conversion of crystalline fenofibrate into the amorphous state by hot melt extrusion for all ternary blends. In contrast, both the binary blends (HPL- and copovidone-based) contained minor remaining crystallites. Irrespectively, all solid dispersions induced during dissolution studies a supersaturated state of FEN, where the ternary ASDs showed enhanced and more complete release of FEN as compared to the binary blends and, even more pronounced, in comparison to the marketed micronized and nano-milled formulations. In terms of the cumulated amount permeated, there were marginal differences between the various formulations when combined dissolution/permeation was done using FeSSIF as donor medium; with FaSSIF as donor medium, the binary HPL-ASD containing the grade with the highest phosphatidylcholine fraction performed best in terms of permeation, even significantly better than the marketed nano-crystal formulation. Otherwise, no significant differences were seen between the various grades of HPL when FEN dissolution and permeation were analyzed for ternary solid dispersions. In conclusion, the in-vitro biopharmaceutical behaviour of hydrogenated phospholipid-containing blends manufactured by hot melt extrusion appears promising. They can be a viable formulation option for poorly water-soluble and lipophilic drug compounds like FEN., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Purpose: Breast cancer is a complex disease with several molecular subtypes that respond differently to therapy. This paper describes liposomes loaded with gold nanoparticles as a targeted drug delivery method in the rapidly developing precision breast cancer treatment area. The aim was to investigate the cytotoxicity level and cellular uptake using several breast cancer cell lines and a normal breast cell line., Materials and Methods: We synthesized gold nanoparticles incorporated in liposomes. Nanostructures were incubated with breast cancer cell lines of different subtypes. The analysis included MTT assay, flow cytometry and immunofluorescence., Results: Cell viability varied among different cancer cells. Moreover, the time- and concentration-dependent manner of viability change was observed. The internalization of liposomes with gold nanoparticles and nanoparticles alone determined different results depending on molecular breast cancer subtypes. The luminal B and triple-negative breast cancer cells demonstrated the highest resistance and sensitivity, respectively. The intensity of cells' interaction with the proposed nanostructures was observed in both cell lines. In this study, we compare the molecular subtypes of breast cancer and discuss how this novel method might improve the therapy success., Conclusions: Our research sheds light on the possibility of new individualized treatments for breast cancer patients, opening the path for better results and a more detailed cancer therapy strategy., Competing Interests: Declaration of competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Amorphous solid dispersions (ASD) represent a viable formulation strategy to improve dissolution and bioavailability of poorly soluble drugs. Our study aimed to evaluate the feasibility and potential role of hydrogenated phospholipid (HPL) as a matrix material and solubilizing additive for binary (alone) or ternary (in combination with polymers) solid dispersions, using fenofibrate (FEN) as the model drug. FEN, incorporated within ASDs by melting or freeze-drying (up to 20% m/m), stayed amorphous during short-term stability studies. The solubility enhancing potential of HPL depended on the dissolution medium. In terms of enhancing in vitro permeation, solid dispersions with HPL were found equally or slightly more potent as compared to the polymer-based ASD. For studied ASD, in vitro permeation was found substantially enhanced as compared to a suspension of crystalline FEN and at least equal compared to marketed formulations under comparable conditions (literature data). Additionally, while the permeation of neat FEN and FEN in binary solid dispersions was affected by the dissolution medium (i.e., the "prandial state"), for ternary solid dispersions the permeation was independent of the "prandial state" (FaSSIF = FeSSIF). This suggests that ternary solid dispersions containing both polymer and HPL may represent a viable formulation strategy to mitigate fenofibrate's food effect., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Paulina Skupin-Mrugalska reports financial support was provided by Phospholipid Research Center. Mikolaj Czajkowski reports financial support was provided by Phospholipid Research Center. Martin Brandl reports a relationship with Phospholipid Research Center that includes: funding grants. Ann-Christin Jacobsen reports a relationship with Phospholipid Research Center that includes: funding grants. Paulina Skupin-Mrugalska reports a relationship with APV Mainz that includes: travel reimbursement. Martin Brandl reports a relationship with APV Mainz that includes: travel reimbursement. Ann-Christin Jacobsen reports a relationship with APV Mainz that includes: travel reimbursement. Annette Bauer-Brandl reports a relationship with APV Mainz that includes: travel reimbursement. Martin Brandl reports a relationship with University of Maryland Baltimore that includes: travel reimbursement. Annette Bauer-Brandl reports a relationship with University of Maryland Baltimore that includes: travel reimbursement. Annette Bauer-Brandl has patent PermeaPad patent owned by SDU licensed to out-licensed to InnoMe.]., (Copyright © 2023. Published by Elsevier B.V.)

3'-hydroxy-3,4,5,4'-tetramethoxystilbene (DMU-214) belongs to methoxystilbenes family and is an active metabolite of 3,4,5,4'-tetramethoxystilbene (DMU-212). In several of our previous studies, the anti-apoptotic activity of DMU-214 was significantly higher than that of the parent compound, especially in ovarian cancer cells. Due to increased lipophilicity and limited solubility, methoxystilbenes require a solubilization strategy enabling DMU-214 administration to the aqueous environment. In this study, DMU-214-loaded liposomes were developed for the first time, and its antitumor activity was tested in the ovarian cancer model.First, several liposomal formulations of DMU-214 were obtained by the thin lipid film hydration method followed by extrusion and then characterized. The diameter of the resulting vesicles was in the range of 118.0-155.5 nm, and samples presented monodisperse size distribution. The release of DMU-214 from the studied liposomes was governed by the contribution of two mechanisms, Fickian diffusion and liposome relaxation.Subsequently, in vitro activity of DMU-214 in the form of a free compound or liposome-bound was studied, including commercial cell line SK-OV-3 and patient-derived ovarian cancer cells in monolayer and spheroid cell culture models. DMU-214 liposomal formulations were found to be more potent (had lower IC 50 values) than the free DMU-214 both in the monolayer and, more significantly, in both examined spheroid models. The above results, with particular emphasis on the patient-derived ovarian cancer model, indicate the importance of further development of liposomal DMU-214 as a potential anticancer formulation for ovarian cancer treatment.

Thymoquinone has been proved to be effective against neoplasms, including skin cancer. Its high lipophilicity, however, may limit its potential use as a drug. Melanoma remains the deadliest of all skin cancers worldwide, due to its high heterogeneity, depending on the stage of the disease. Our goal was to compare the anti-cancer activity of free thymoquinone and thymoquinone-loaded liposomes on two melanoma cell lines that originated from different stages of this cancer: skin-derived A375 and metastatic WM9. We evaluated the proapoptotic effects of free thymoquinone by flow cytometry and Western blot, and its mitotoxicity by means of JC-1 assay. Additionally, we compared the cytotoxicity of free thymoquinone and thymoquinone in liposomes by WST-1 assay. Our results revealed a higher antiproliferative effect of TQ in WM9 cells, whereas its higher proapoptotic activity was observed in the A375 cell line. Moreover, the thymoquinone-loaded liposome was proved to exert stronger cytotoxic effect on both cell lines studied than free thymoquinone. Differences in the response of melanoma cells derived from different stages of the disease to thymoquinone, as well as their different responses to free and carrier-delivered thymoquinone, are essential for the development of new anti-melanoma therapies. However, further research is required to fully understand them.

Perindopril arginine (PA) as an angiotensin-converting enzyme (ACE) inhibitor is widely used in cardiovascular diseases, especially in systemic hypertension and heart failure. Although the pharmacokinetics of PA are well documented, there is no available detailed data on its permeation in in vitro conditions. The present study aimed to assess the transport of PA across both biological membranes and artificial biomimetic ones. For the determination of PA transport, the Caco-2 cell line was selected as a reliable in vitro model of gastrointestinal biological barriers. Additionally, a novel 96-well plate with phospholipid membrane PermeaPad was used to evaluate the transport of PA by passive diffusion. We confirmed that PA is relatively poorly permeable across the Caco-2 monolayer. The permeability results obtained from the non-cell-based model demonstrated higher transport of PA as compared to that of Caco-2. Thus, PA transport across the biological membranes might be suggested to be regulated by the membrane transporters.

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.

Liposomes are phospholipid-based self-assembled nanoparticles. Various components can be solubilized in the lipid bilayer, encapsulated in the aqueous core or attached to the surface, making liposomes attractive platforms for multimodality functionalization. Here we describe theranostic liposomes delivering a magnetic resonance contrast agent (lipid derivative of gadopentetic acid) and a hydrophobic photosensitizer (zinc phthalocyanine, ZnPc) for photodynamic therapy of cancer. For the first time, this theranostic system was prepared by the microfluidic method. Analogous formulations were produced by thin lipid film hydration (TLH) with down-sizing performed by extrusion for comparison purposes. We demonstrated double the loading capacity of ZnPc into liposomes made by microfluidics compared to TLH/extrusion. Microfluidics resulted in the theranostic nanoliposomes characterized by sizes =2.5x smaller than vesicles prepared by TLH/extrusion. Increased relaxivity was observed for liposomes manufactured by microfluidics compared to TLH, despite a slightly lower Gd chelate recovery. We attributed the improved relaxation to the increased surface area/volume ratio of vesicles and decreased phosphatidylcholine/ZnPc molar ratio, which affected water molecules' diffusion through the liposomal membrane. Finally, we showed photodynamic efficacy of ZnPc loaded into theranostic liposomes in head and neck cancer model, resulting in IC 50 of 0.22 - 0.61 μM, depending on the formulation and cell line used. We demonstrate microfluidics' feasibility to be used for theranostic liposome manufacturing and co-entrapment of therapeutic and imaging components in a single-step process with a high yield., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Despite the increasing development of medicine, ovarian cancer is still a high-risk, metastatic disease that is often diagnosed at a late stage. In addition, difficulties in its treatment are associated with high resistance to chemotherapy and frequent relapse. Cancer stem cells (CSCs), recently attracting significant scientific interest, are considered to be responsible for the malignant features of tumors. CSCs, as the driving force behind tumor development, generate new cells by modifying different signaling pathways. Moreover, investigations on different types of tumors have shown that signaling pathways are key to epithelial-mesenchymal transition (EMT) regulation, metastasis, and self-renewal of CSCs. Based on these established issues, new therapies are being investigated based on the use of inhibitors to block CSC growth and proliferation signals. Many reports indicate that CSC markers play a key role in cancer metastasis, with hopes placed in their targeting to block this process and eliminate relapses. Current histological classification of ovarian tumors, their epidemiology, and the most recent knowledge of ovarian CSCs, with particular emphasis on their molecular background, are important aspects for consideration. Furthermore, the importance of signaling pathways involved in tumor growth, development, and metastasis, is also presented.

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.

Resveratrol is a phytoalexin that naturally occurs in grapes, blueberries, cranberries, peanuts and many other plants. Although resveratrol inhibits carcinogenesis in all three stages, its clinical application is restricted due to poor pharmacokinetics. The methylated analogues of resveratrol have been found to have higher bioavailability and cytotoxic activity than that of the prototupe compound. Among the various methoxy derivatives of resveratrol, 3,4,5,4'-tetrametoxystilbene (DMU-212) is suggested to be one of the strongest activators of cytotoxicity and apoptosis. DMU-212 has been shown to exert anti-tumor activity in DLD-1 and LOVO colon cancer cells. Since colorectal cancer is the third most common cause of cancer-related deaths worldwide, the development of new anticancer agents is nowadays of high significance. The aim of the present study was to assess the anticancer activity of 4'-hydroxy-3,4,5-trimetoxystilbene (DMU-281), the metabolite of DMU-212, in DLD-1 and LOVO cell lines. We showed for the first time the cytotoxic activity of DMU-281 triggered via cell cycle arrest at G2/M phase and apoptosis induction accompanied by the activation of caspases-9, -8, -3/7. Furthermore, DMU-281 has been found to change the expression pattern of genes and proteins related to intrinsic as well as extrinsic apoptosis. Since the activation of these pathways of apoptosis is still the most desired strategy in anticancer research, DMU-281 seems to provide a promising approach to the treatment of colon cancer.

Targeting tumor cell motility and proliferation is an extremely important challenge in the prevention of metastasis and improving the effectiveness of cancer treatment. We recently published data revealing that DMU-214, the metabolite of firmly cytotoxic resveratrol analogue DMU-212, exerted significantly higher biological activity than the parent compound in ovarian cancer cells. The aim of the present study was to assess the molecular mechanism of the potential anti-migration and anti-proliferative effect of DMU-214 in ovarian cancer cell line SKOV-3. We showed that DMU-214 reduced the migratory capacity of SKOV-3 cells. The microarray analysis indicated ontology groups of genes involved in processes of negative regulation of cell motility and proliferation. Furthermore, we found DMU-214 triggered changes in expression of several migration- and proliferation-related genes (SMAD7, THBS1, IGFBP3, KLF4, Il6, ILA, SOX4, IL15, SRF, RGCC, GPR56) and proteins (GPR56, RGCC, SRF, SMAD7, THBS1), which have been shown to interact to each other to reduce cell proliferation and motility. Our study showed for the first time that DMU-214 displayed anti-migratory and anti-proliferative activity in SKOV-3 ovarian cancer cells. On the basis of whole transcriptome analysis of these cells, we provide new insight into the role of DMU-214 in inhibition of processes related to metastasis.

The multi-drug resistant bacteria have become a serious problem complicating therapies to such a degree that often the term "post-antibiotic era" is applied to describe the situation. The infections with methicillin-resistant S. aureus, vancomycin-resistant E. faecium, third generation cephalosporin-resistant E. coli, third generation cephalosporin-resistant K. pneumoniae and carbapenem-resistant P. aeruginosa have become commonplace. Thus, the new strategies of infection treatment have been searched for, and one of the approaches is based on photodynamic antimicrobial chemotherapy. Photodynamic protocols require the interaction of photosensitizer, molecular oxygen and light. The aim of this review is to provide a comprehensive overview of photodynamic antimicrobial chemotherapy by porphyrinoid photosensitizers. In the first part of the review information on the mechanism of photodynamic action and the mechanism of the bacteria resistance to the photodynamic technique were described. In the second one, it was described porphyrinoids photosensitizers like: porphyrins, chlorins and phthalocyanines useable in photodynamic bacteria inactivation., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

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 IC 50 values. The lowest IC 50 s (7-13 nM) were obtained with a PS encapsulated in cationic liposomes.

The present study is focused on the development of liposomes bearing gadolinium chelate (GdLip) providing two functionalities for magnetic resonance imaging (MRI) and photodynamic therapy of cancer. A lipid derivative of gadolinium(III) diethylenetriamine pentaacetic acid salt (GdDTPA1) was inserted in the liposomal membrane and served as MRI contrast agent whereas a zinc phthalocyanine (ZnPc) was used as a model photosensitizer. In addition to conventional liposomes, pegylated lipids were used for the preparation of "stealth" liposomes. The characterization of different GdLip formulations involved evaluation of the liposomes size by nanoparticle tracking analysis, thermal phase behavior by differential scanning calorimetry and ZnPc-mediated singlet oxygen production. Furthermore, relaxivity measurements were performed as well as cytotoxicity and photodynamic activity against cancerous and normal cell lines was studied. Size and thermal behavior were only slightly influenced by GdLip composition, however it distinctly affected singlet oxygen production of ZnPc-loaded GdLip. The quantum yields of singlet oxygen generation by zinc phthalocyanine incorporated in GdLip containing cationic or/and pegylated lipids were smaller than those obtained for non-pegylated carriers with l-α-phosphatidylglycerol. In general, all formulations of GdLip, irrespectively of composition, were characterized by relaxivities higher than those of commercially used contrast agents (e.g. Magnevist®). NMR study has shown that the incorporation of ZnPc into the formulations of GdLip increases the relaxation parameters r 1 and r 2 , compared to the values for the non-loaded vesicles. GdDTPA1 did not influence the photodynamic activity of ZnPc against HeLa cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Photodynamic therapy involves the use of a photosensitizer that is irradiated with visible light in the presence of oxygen, resulting in the formation of reactive oxygen species. A novel phthalocyanine derivative, the quaternary iodide salt of magnesium(II) phthalocyanine with N-methyl morpholiniumethoxy substituents, was synthesized, and characterized. The techniques used included mass spectrometry (MALDI TOF), UV-vis, NMR spectroscopy, and photocytotoxicity against bacteria, fungi and cancer cells. The phthalocyanine derivative possessed typical characteristics of compounds of the phthalocyanine family but the effect of quaternization was observed on the optical properties, especially in terms of absorption efficiency. The results of the photodynamic antimicrobial effect study demonstrated that cationic phthalocyanine possesses excellent photodynamic activity against planktonic cells of both Gram-positive and Gram-negative bacteria. The bactericidal effect was dose-dependent and all bacterial strains tested were killed to a significant degree by irradiated phthalocyanine at a concentration of 1×10 -4 M. There were no significant differences in the susceptibility of Gram-positive and Gram-negative bacteria to the applied photosensitizer. The photosensitivity of bacteria in the biofilm was lower than that in planktonic form. No correlation was found between the degree of biofilm formation and susceptibility to antimicrobial photodynamic inactivation. The anticancer activity of the novel phthalocyanine derivative was tested using A549 adenocarcinomic alveolar basal epithelial cells and the human oral squamous cell carcinoma cells derived from tongue (HSC3) or buccal mucosa (H413). No significant decrease in cell viability was observed under different conditions or with different formulations of the compound., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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.

Four novel magnesium(II) and zinc(II) phthalocyanines bearing 1,4,7-trioxanonyl, polyether and/or (2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy, heterocyclic substituents at their non-peripheral positions were synthesized and assessed in terms of physicochemical and biological properties. Magnesium phthalocyanine derivatives bearing polyether substituents (Pc-1), a mixed system of polyether and heterocyclic substituents (Pc-3), and four heterocyclic substituents (Pc-4), respectively, were synthesized following the Linstead macrocyclization reaction procedure. Zinc phthalocyanine (Pc-2) bearing polyether substituents at non-peripheral positions was synthesized following the procedure in n-pentanol with the zinc acetate, and DBU. Novel phthalocyanines were purified by flash column chromatography and characterized using NMR, MS, UV-Vis and HPLC. Moreover, two precursors in macrocyclization reaction phthalonitriles were characterized using X-ray. Photophysical properties of the novel macrocycles were evaluated, including UV-Vis spectra analysis and aggregation study. All macrocycles subjected to singlet oxygen generation and the oxidation rate constant measurements exhibited lower quantum yields of singlet oxygen generation in DMSO than in DMF. In addition, the Pc-2 molecule was found to be the most efficient singlet oxygen generator from the group of macrocycles studied. The photocytotoxicity evaluated on the human oral squamous cell carcinoma cell line, HSC-3, for Pc-3 was significantly higher than that for Pc-1, Pc-2, and Pc-4. Interestingly, Pc-3 was found to be the most active macrocycle in vitro although its ability to generate singlet oxygen was significantly lower than those of Pc-1 and Pc-2. However, attempts to encapsulate phthalocyanines Pc-1-Pc-3 in liposomal membranes were unsuccessful. The phthalocyanine-nitroimidazole conjugate, Pc-4 was encapsulated in phosphatidylglycerol:phosphatidylcholine unilamellar liposomes and subjected to photocytotoxicity study., (© 2013.)

The complete eradication of various targets, such as infectious agents or cancer cells, while leaving healthy host cells untouched, is still a great challenge faced in the field of medicine. Photodynamic therapy (PDT) seems to be a promising approach for anticancer treatment, as well as to combat various dermatologic and ophthalmic diseases and microbial infections. The application of liposomes as delivery systems for porphyrinoids has helped overcome many drawbacks of conventional photosensitizers and facilitated the development of novel effective photosensitizers that can be encapsulated in liposomes. The development, preclinical studies and future directions for liposomal delivery of conventional and novel photosensitizers are reviewed., (Copyright © 2013 Elsevier Ltd. All rights reserved.)