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The most important goal of regenerative medicine is to repair, restore, and regenerate tissues and organs that have been damaged as a result of an injury, congenital defect or disease, as well as reversing the aging process of the body by utilizing its natural healing potential. Regenerative medicine utilizes products of cell therapy, as well as biomedical or tissue engineering, and is a huge field for development. In regenerative medicine, stem cells and growth factor are mainly used; thus, innovative drug delivery technologies are being studied for improved delivery. Drug delivery systems offer the protection of therapeutic proteins and peptides against proteolytic degradation where controlled delivery is achievable. Similarly, the delivery systems in combination with stem cells offer improvement of cell survival, differentiation, and engraftment. The present review summarizes the significance of biomaterials in tissue engineering and the importance of colloidal drug delivery systems in providing cells with a local environment that enables them to proliferate and differentiate efficiently, resulting in successful tissue regeneration.

Achievement of a high dose of drug in the tumor while minimizing its systemic side effects is one of the important features of an improved drug delivery system. Thus, developing responsive carriers for site-specific delivery of chemotherapeutic agents has become a main goal of research efforts. One of the known hallmarks of cancerous tumors is hypoxia, which offers a target for selective drug delivery. The stimuli-sensitive micellar system developed by us, (PEG-azobenzene-PEI-DOPE (PAPD) has proven to be effective in vitro. The proposed construct developed, PAPD, contains an azobenzene group as a hypoxia-sensitive moiety that triggers the shedding of the PEG layer from the nanoparticle surface under conditions of hypoxia to improve cellular uptake. Using microfluidics, we show significantly improved cellular association and penetration under hypoxia in both single cells and in a 3D tumor model. Employing an in vivo model, we demonstrate slower tumor growth that did not induce systemic side effects, including weight loss in an experimental animal model, when compared to the free drug treatment. This complex-in-nature but simple-in-design system for the simultaneous delivery of siRNA to silence the P-glycoprotein and doxorubicin with active tumor targeting and proven therapeutic efficacy represents a universal platform for the delivery of other hydrophobic chemotherapeutic agents and siRNA molecules which can be further modified.

Amphotericin B (AmB) belongs to a group of polyene antibiotics commonly used in the treatment of systemic mycotic infections. A widely accepted mechanism of action of AmB is based on the formation of an oligomeric pore structure within the plasma membrane (PM) by interaction with membrane sterols. Although AmB binds preferentially to ergosterol, it can also bind to cholesterol in the mammalian PM and cause severe cellular toxicity. The lipid content and its lateral organization at the cell PM appear to be significant for AmB binding. Several ATP-binding cassette (ABC) transporters, including ABCA1, play a crucial role in lipid translocation, cholesterol redistribution and efflux. Here, we demonstrate that cells expressing ABCA1 are more resistant to AmB treatment, while cells lacking ABCA1 expression or expressing non-active ABCA1MM mutant display increased sensitivity. Further, a FLIM analysis of AmB-treated cells reveals a fraction of the antibiotic molecules, characterized by relatively high fluorescence lifetimes (> 6 ns), involved in formation of bulk cholesterol–AmB structures at the surface of ABCA1-expressing cells. Finally, lowering the cellular cholesterol content abolishes resistance of ABCA1-expressing cells to AmB. Therefore, we propose that ABCA1-mediated cholesterol efflux from cells induces formation of bulk cholesterol–AmB structures at the cell surface, preventing AmB cytotoxicity. Electronic supplementary material The online version of this article (10.1007/s00018-019-03154-w) contains supplementary material, which is available to authorized users.

Dendrimers comprise a specific group of macromolecules, which combine structural properties of both single molecules and long expanded polymers. The three-dimensional form of dendrimers and the extensive possibilities for use of additional substrates for their construction creates a multivalent potential and a wide possibility for medical, diagnostic and environmental purposes. Depending on their composition and structure, dendrimers have been of interest in many fields of science, ranging from chemistry, biotechnology to biochemical applications. These compounds have found wide application from the production of catalysts for their use as antibacterial, antifungal and antiviral agents. Of particular interest are peptide dendrimers as a medium for transport of therapeutic substances: synthetic vaccines against parasites, bacteria and viruses, contrast agents used in MRI, antibodies and genetic material. This review focuses on the description of the current classes of dendrimers, the methodology for their synthesis and briefly drawbacks of their properties and their use as potential therapies against infectious diseases.

The liposomes have continued to be well-recognized as an important nano-sized drug delivery system with attractive properties, such a characteristic bilayer structure assembling the cellular membrane, easy-to-prepare and high bio-compatibility. Extensive effort has been devoted to the development of liposome-based drug delivery systems during the past few decades. Many drug candidates have been encapsulated in liposomes and investigated for reduced toxicity and extended duration of therapeutic effect. The liposomal encapsulation of hydrophilic and hydrophobic small molecule therapeutics as well as other large molecule biologics have been established among different academic and industrial research groups. To date, there has been an increasing number of FDA-approved liposomal-based therapeutics together with more and more undergoing clinical trials, which involve a wide range of applications in anticancer, antibacterial, and antiviral therapies. In order to meet the continuing demand for new drugs in clinics, more recent advancements have been investigated for optimizing liposomal-based drug delivery system with more reproducible preparation technique and a broadened application to novel modalities, including nucleic acid therapies, CRISPR/Cas9 therapies and immunotherapies. This review focuses on the recent liposome' preparation techniques, the excipients of liposomal formulations used in various novel studies and the routes of administration used to deliver liposomes to targeted areas of disease. It aims to update the research in liposomal delivery and highlights future nanotechnological approaches.

Neutrophils can release DNA and granular cytoplasmic proteins that form smooth filaments of stacked nucleosomes (NS). These structures, called neutrophil extracellular traps (NETs), are involved in multiple pathological processes, and NET formation and removal are clinically significant. The monoclonal antibody 2C5 has strong specificity toward intact NS but not to individual NS components, indicating that 2C5 could potentially target NS in NETs. In this study, NETs were generated in vitro using neutrophils and HL-60 cells differentiated into granulocyte-like cells. The specificity of 2C5 toward NETs was evaluated by ELISA, which showed that it binds to NETs with the specificity similar to that for purified nucleohistone substrate. Immunofluorescence showed that 2C5 stains NETs in both static and perfused microfluidic cell cultures, even after NET compaction. Modification of liposomes with 2C5 dramatically enhanced liposome association with NETs. Our results suggest that 2C5 could be used to identify and visualize NETs and serve as a ligand for NET-targeted diagnostics and therapies.

Lipid-polymer hybrid nanoparticles (LPHNP) are delivery systems for controlled drug delivery at tumor sites. The superior biocompatible properties of lipids and structural advantages of polymers can be obtained using this system for controlled drug delivery. In this study, cisplatin-loaded lipid-chitosan hybrid nanoparticles were formulated by the single step ionic gelation method based on ionic interaction of positively charged chitosan and negatively charged lipid. Formulations with various chitosan to lipid ratios were investigated to obtain the optimal particle size, encapsulation efficiency, and controlled release pattern. Transmission electron microscope and dynamic light scattering analysis demonstrated a size range of 181–245 nm and a zeta potential range of 20–30 mV. The stability of the formulation was demonstrated by thermal studies. Cytotoxicity and cellular interaction of cisplatin-loaded LPHNP were investigated using in vitro cell-based assays using the A2780 ovarian carcinoma cell line. The pharmacokinetics study in rabbits supported a controlled delivery of cisplatin with enhanced mean residence time and half-life. These studies suggest that cisplatin loaded LPHNP have promise as a platform for controlled delivery of cisplatin in cancer therapy.

Multiple factors are involved in the development of cancers and their effects on survival rate. Many are related to chemo-resistance of tumor cells. Thus, treatment with a single therapeutic agent is often inadequate for successful cancer therapy. Ideally, combination therapy inhibits tumor growth through multiple pathways by enhancing the performance of each individual therapy, often resulting in a synergistic effect. Polymeric nanoparticles prepared from block co-polymers have been a popular platform for co-delivery of combinations of drugs associated with the multiple functional compartments within such nanoparticles. Various polymeric nanoparticles have been applied to achieve enhanced therapeutic efficacy in cancer therapy. However, reported drug ratios used in such systems often vary widely. Thus, the same combination of drugs may result in very different therapeutic outcomes. In this review, we investigated polymeric co-delivery systems used in cancer treatment and the drug combinations used in these systems for synergistic anti-cancer effect. Development of polymeric co-delivery systems for a maximized therapeutic effect requires a deeper understanding of the optimal ratio among therapeutic agents and the natural heterogenicity of tumors.

Multidrug resistance (MDR) significantly decreases the therapeutic efficiency of anti-cancer drugs. Its reversal could serve as a potential method to restore the chemotherapeutic efficiency. Downregulation of MDR-related proteins with a small interfering RNA (siRNA) is a promising way to reverse the MDR effect. Additionally, delivery of small molecule therapeutics simultaneously with siRNA can enhance the efficiency of chemotherapy by dual action in MDR cell lines. Here, we conjugated the dendrimer, generation 4 polyamidoamine (G4 PAMAM), with a polyethylene glycol (PEG)-phospholipid copolymer. The amphiphilic conjugates obtained spontaneously self-assembled into a micellar nano-preparation, which can be co-loaded with siRNA onto PAMAM moieties and sparingly water-soluble chemotherapeutics into the lipid hydrophobic core. This system was co-loaded with doxorubicin (DOX) and therapeutic siRNA (siMDR-1) and tested for cytotoxicity against MDR cancer cells: human ovarian carcinoma (A2780 ADR) and breast cancer (MCF7 ADR). The combination nanopreparation effectively downregulated P-gp in MDR cancer cells and reversed the resistance towards DOX.

p53, The tumour suppressor protein encoded by P53 gene, is the most commonly altered protein in the human malignancies. MDM2 controls the p53 activity through an autoregulatory feedback loop. p53 activates the expression of MDM2 and in return, MDM2 blocks the p53 activity through various mechanisms. Nutlins, including nutlin-3, are a new class of small molecules that bind to MDM2 and prevent its interaction with p53. This antagonism results in increased p53 activity and can also re-activates the p53 pathway and resensitize the glioblastoma cells to apoptosis. Here we used nutlin-3 in combination with another potent anticancer drug, doxorubicin, to investigate the synergism between these drugs. We encapsulated both water-insoluble drugs in the PEG-PE-based micellar nanocarriers efficiently and evaluate their efficacy against U87MG cells in 2 D and 3 D models. These nanomedicine formulations successfully re-activated the p53 levels in cells, increased the apoptosis and showed strong synergistic cytotoxic effect.

Cancer causes the second-highest rate of death world-wide. A major shortcoming inherent in most of anticancer drugs is their lack of tumor selectivity. Nanodrugs for cancer therapy administered intravenously escape renal clearance, are unable to penetrate through tight endothelial junctions of normal blood vessels and remain at a high level in plasma. Over time, the concentration of nanodrugs builds up in tumors due to the EPR effect, reaching several times higher than that of plasma due to the lack of lymphatic drainage. This review will address in detail the progress and prospects of tumor-targeting via EPR effect for cancer therapy.

Recently, it has been discovered that the PEG layer on nanoparticle surface can create steric hindrance, preventing efficient cellular uptake of PEGylated nanoparticles. Thus, it would be ideal to have a nanoparticle system that sheds the PEG layer upon reaching the tumor microenvironment. Hypoxia, which is a hallmark of cancerous tumors, can be used as a trigger to shed the PEG layer from the nanoparticle surface. In this study, a hypoxia-sensitive PEG-azobenzene-PEI-DOPE (PAPD) construct, with an azobenzene group as a hypoxia-sensitive moiety, was prepared. The feasibility of co-delivering Doxorubicin (Dox) and anti-P-gp siRNA (siPgp) using the PAPD nanoparticles was evaluated in monolayers of the Adriamycin-resistant human ovarian cancer cell line, A2780 ADR, and in 3D spheroids of the multidrug-resistant human breast cancer cell line, MCF7 ADR. Under hypoxic conditions, the PAPD nanoparticles showed up to a 60% increase in cellular uptake by monolayers and a significantly greater tumor penetration in a spheroid model. siPgp, when delivered using PAPD nanoparticles, showed up to a 60% P-gp downregulation under hypoxic conditions. The combination of siPgp and Dox delivered using PAPD nanoparticles led to an 80% cytotoxicity in cell monolayers and 20% cytotoxicity in spheroids under hypoxic conditions. In this research, a novel hypoxia-sensitive nanoparticle system was developed that demonstrated improved delivery of an encapsulated cargo and augmented cytotoxicity on multidrug-resistant cancer cells under hypoxic conditions.

Folic acid is often used for active targeting of tumor cells to enhance therapeutic outcomes. Here, folic acid was conjugated with chitosan and folate-conjugated chitosan-lipid hybrid nanoparticles were prepared by ionic gelation method using anionic lipid. These nanoparticles were in size range of 200 to 400 nm with spherical shape. In vitro drug release data suggested a sustained release of cisplatin. The therapeutic efficacy of the folate-conjugated hybrid nanoparticles was evaluated in SK-OV-3, A2780 and MCF-7 cancer cell lines. A significant increase in cytotoxicity was observed with folate targeted LPHNPs compared to non-targeted LPHNPs. Significantly enhanced cellular uptake and cell cycle arrest resulting from folate-targeted nanoparticles were confirmed using fluorescence microscopy and flow cytometry. The therapeutic efficacy and tumor penetration were further evaluated in 3D spheroid tumor models. These studies suggest that folate-conjugated lipid-chitosan nanoparticles could enhance therapeutic activity and may represent a promising platform for active targeting of tumor cells.

A series of caffeic acid derivatives were synthesized via a modified Wittig reaction which is a very important tool in organic chemistry for the construction of unsaturated carbon–carbon bonds. All reactions were performed in water medium at 90 °C. The aqueous Wittig reaction worked best when one unprotected hydroxyl group was present in the phenyl ring. The olefinations in the aqueous conditions were also conducted with good yields in the presence of two unprotected hydroxyl groups. When the number of the hydroxyl groups was increased to three, the reaction yields were worse, and the derivatives 12, 13, and 18 were obtained with 74%, 37%, and 70% yields, respectively. Nevertheless, the Wittig reaction using water as the essential medium is an elegant one-pot synthesis and a greener method, which can be a safe alternative for implementation in organic chemistry. The obtained compounds were tested for their antioxidant activity, and 12, 13, and 18 showed the highest activities. Moreover, all synthesized compounds displayed no cytotoxicity, and can therefore be used in the pharmaceutical or cosmetic industry.

The effect of existing anti-cancer therapies is based mainly on the stimulation of apoptosis in cancer cells. Here, we have demonstrated the ability of a catalytically-reactive nanoparticle-based complex of cytochrome c with cardiolipin (Cyt-CL) to induce the apoptosis and killing of cancer cells in a monolayer cell culture. Cyt-CL nanoparticles were prepared by complexing CytC with different molar excesses of CL. Following characterization, cytotoxicity and apoptosis inducing effects of nanoparticles were investigated. In an attempt to identify the anticancer activity mechanism of Cyt-CL, pseudo-lipoxygenase and lipoperoxidase reaction kinetics were measured by chemiluminescence. Using chemiluminescence, we have demonstrated that the Cyt-CL complex produces lipoperoxide radicals in two reactions: by decomposition of lipid hydroperoxides, and by lipid peroxidation under the action of H2O2. Antioxidants inhibited the formation of lipid radicals. Cyt-CL nanoparticles, but not the CytC alone, dramatically enhanced the level of apoptosis and cell death in two cell lines: drug-sensitive (A2780) and doxorubicin-resistant (A2780-Adr). The proposed mechanism of the cytotoxic action of Cyt-CL involves either penetration through the cytoplasm and outer mitochondrial membrane and catalysis of lipid peroxidation reactions at the inner mitochondrial membrane, or/and activation of lipid peroxidation within the cytoplasmic membrane. Here we propose a new type of anticancer nano-formulation, with an action based on the catalytic action of Cyt-CL nanoparticles on the cell membrane and and/or mitochondrial membranes that results in lipid peroxidation reactions, which give rise to activation of apoptosis in cancer cells, including multidrug resistant cells.

Mateusz Legut, Dominik Lipka, Nina Filipczak, Adriana Piwoni, Arkadiusz Kozubek, Jerzy GubernatorDepartment of Lipids and Liposomes, Faculty of Biotechnology, University of Wrocław, Wrocław, PolandAbstract: This paper describes a novel formulation of antineoplastic drug: mitoxantrone loaded into liposomal carriers enriched with encapsulated anacardic acid in the liposomal bilayer using a vitamin C gradient. Anacardic acid is a potent epigenetic agent with anticancer activity. This is the first liposomal formulation to combine an actively encapsulated drug and anacardic acid. The liposomes were characterized in terms of basic parameters, such as size, zeta potential, optimal drug-to-lipid ratio, loading time and temperature, and stability at 4°C and in human plasma in vitro. The formulation was found to be stable, and the loading process was rapid and efficient (drug-to-lipid ratio of up to 0.3 with over 90% efficiency in 5 minutes). The cytotoxicity of these formulations was assessed using the human melanoma cell lines A375 and Hs294T and the normal human dermal fibroblast line. The results showed that anacardic acid and to a smaller extent vitamin C significantly increased the cytotoxicity of the drug towards melanoma compared to ammonium sulfate liposomes. On the other hand, vitamin C and anacardic acid both protected normal cells from damage caused by the drug. The formulation combining anacardic acid, vitamin C, and mitoxantrone showed promising results in terms of cytotoxicity and cytoprotection. Therefore, it has potential for anticancer treatment.Keywords: anacardic acid, vitamin C, ascorbic acid, liposomes, mitoxantrone, melanomaCorrigendum for this paper has been published&nbsp

The incorporation of hydrophobic drugs into liposomes improve their bioavailability and leads to increased stability and anticancer activity, along with decreased drug toxicity. Curcumin (Cur) is a natural polyphenol compound with a potent anticancer activity in pancreatic adenocarcinoma (PA). In the present study, different types of Cur-loaded liposomal formulations were prepared and characterized in terms of size, shape, zeta potential, optimal drug-to-lipid ratio and stability at 4°C, 37°C; and in human plasma in vitro. The best formulation in terms of these parameters was PEGylated, cholesterol-free formulation based upon hydrogenated soya PC (HSPC:DSPE-PEG2000:Cur, termed H5), which had a 0.05/10 molar ratio of drug-to-lipid, was found to be stable and had a 96% Cur incorporation efficiency. All Cur-loaded liposomal formulations had potent anticancer activity on the PA cancer cell lines AsPC-1 and BxPC-3, and were less toxic to a normal cell line (NHDF). Furthermore, apoptosis-induction induced by Cur in PA cells was associated with morphological changes including cell shrinkage, cytoplasmic blebbing, irregularity in shape and the externalization of cell membrane phosphatidylserine, which was preceded by an increase in intracellular reactive oxygen species (ROS) generation and caspase 3/7 activation. Because the liposomal formulations tested here, especially the H5 variant which exhibited slow release of the Cur in the human plasma test, the formulation may be stable enough to facilitate the accumulation of pharmacologically active amounts of Cur in target cancer tissue by EPR. Therefore, our formulations could serve as a promising therapeutic approach for pancreatic cancer and other cancers.

Dominik Lipka,1 Jerzy Gubernator,1 Nina Filipczak,1 Sabine Barnert,2 Regine Süss,2 Mateusz Legut,1 Arkadiusz Kozubek1 1Department of Lipids and Liposomes, University of Wroclaw, Wroclaw, Poland; 2Department of Pharmaceutical Technology, Albert Ludwigs University, Freiburg, Germany Abstract: The encapsulation of anticancer drugs in a liposome structure protects the drug during circulation and increases drug accumulation in the cancer tissue and antitumor activity while decreasing drug toxicity. This paper presents a new method of active drug loading based on a vitamin C pH/ion gradient. Formulations were characterized in terms of the following parameters: optimal external pH, time and drug-to-lipid ratio for the purpose of remote loading, and in vitro stability. In the case of the selected drug, epirubicin (EPI), its coencapsulation increases its anticancer activity through a possibly synergistic effect previously reported by other groups for a free nonencapsulated drug/vitamin C cocktail. The method also has another advantage over other remote-loading methods: it allows faster drug release through liposome destabilization at the tumor site, thanks to the very good solubility of the EPI vitamin C salt, as seen on cryogenic transmission electron microscopy images. This influences the drug-release process and increases the anticancer activity of the liposome formulation. The liposomes are characterized as stable, with very good pharmacokinetics (half-life 18.6 hours). The antitumor activity toward MCF-7 and 4T-1 breast cancer cells was higher in the case of EPI loaded via our gradient than via an ammonium sulfate gradient. Finally, the EPI liposomal formulation and the free drug were tested using the murine 4T-1 breast cancer model. The antitumor activity of the encapsulated drug was confirmed (tumor-growth inhibition over 40% from day 16 until the end of the experiment), and the free drug was shown to have no anticancer activity at the tested dose. Keywords: liposomes, epirubicin, vitamin C, antitumor activity, remote loading, ascorbic acid