Solvent-Free Synthesis of Multifunctional Block Copolymer and Formation of DNA and Drug Nanocarriers.

The synthesis of well-defined multifunctional polymers is of great importance for the development of complex materials for biomedical applications. In the current work, novel and multi-amino-functional diblock copolymer for potential gene and drug delivery applications was successfully synthesized. A highly efficient one-step and quantitative modification of an alkyne-functional polycarbonate-based precursor was performed, yielding double hydrophilic block copolymer with densely grafted primary amine side groups. The obtained positively charged block copolymer co-associated with DNA, forming stable and biocompatible nanosized polyplexes. Furthermore, polyion complex (PIC) micelles with tunable surface charge and decorated with cell targeting moieties were obtained as a result of direct mixing in aqueous media of the multi-amino-functional block copolymer and a previously synthesized oppositely charged block copolymer bearing disaccharide end-group. The obtained well-defined nanosized PIC-micelles were loaded with the hydrophobic drug curcumin. Both types of nanoaggregates (polyplexes and PIC-micelles) were physico-chemically characterized. Moreover, initial in vitro evaluations were performed to assess the nanocarriers' potential for biomedical applications.

Green Synthesis and the Evaluation of a Functional Amphiphilic Block Copolymer as a Micellar Curcumin Delivery System.

Polymer micelles represent one of the most attractive drug delivery systems due to their design flexibility based on a variety of macromolecular synthetic methods. The environmentally safe chemistry in which the use or generation of hazardous materials is minimized has an increasing impact on polymer-based drug delivery nanosystems. In this work, a solvent-free green synthetic procedure was applied for the preparation of an amphiphilic diblock copolymer consisting of biodegradable hydrophobic poly(acetylene-functional carbonate) and biocompatible hydrophilic polyethylene glycol (PEG) blocks. The cyclic functional carbonate monomer 5-methyl-5-propargyloxycarbonyl-1,3-dioxane-2-one (MPC) was polymerized in bulk using methoxy PEG-5K as a macroinitiator by applying the metal-free organocatalyzed controlled ring-opening polymerization at a relatively low temperature of 60 °C. The functional amphiphilic block copolymer self-associated in aqueous media into stable micelles with an average diameter of 44 nm. The copolymer micelles were physico-chemically characterized and loaded with the plant-derived anticancer drug curcumin. Preliminary in vitro evaluations indicate that the functional copolymer micelles are non-toxic and promising candidates for further investigation as nanocarriers for biomedical applications.

Three-Dimensional Nucleic Acid Nanostructures Based on Self-Assembled Polymer-Oligonucleotide Conjugates of Comblike and Coil-Comb Chain Architectures.

Spherical nucleic acids have emerged as a class of nanostructures, exhibiting a wide variety of properties, distinctly different from those of linear nucleic acids, and a plethora of applications in therapeutics and diagnostics. Herein, we report on preparation of 3D nucleic acid nanostructures, prepared by self-assembly of polymer-oligonucleotide conjugates. The latter are obtained by grafting multiple alkyne-functionalized oligonucleotide strands onto azide-modified homo-, block, and random (co)polymers of chloromethylstyrene via initiator-free click coupling chemistry to form conjugates of comblike and coil-comb chain architectures. The resulting conjugates are amphiphilic and form stable nanosized supramolecular structures in aqueous solution. The nanoconstructs are thoroughly investigated and a number of physical characteristics, in particular, molar mass, size, aggregation number, zeta potential, material density, number of oligonucleotide strands per particle, grafting density, and their relation to hallmark properties of spherical nucleic acids - biocompatibility, resistance against DNase I, cellular uptake without the need for transfection agents - are determined.

Nanoarchitectonics of Spherical Nucleic Acids with Biodegradable Polymer Cores: Synthesis and Evaluation.

Spherical nucleic acids (SNAs) have gained significant attention due to their unique properties allowing them to overcome the challenges that face current nanocarriers used for gene therapies. The aim of this study is to synthesize and characterize polymer-oligonucleotide conjugates of different architecture and to evaluate the possibility of forming SNAs with biodegradable cores. Initially, two types of azide (multi)functional polyester-based (co)polymers were successfully synthesized and characterized. In the next step, short oligonucleotide strands were attached to the polymer chains applying the highly efficient and metal-free "click" reaction, thus forming conjugates with block or graft architecture. Both conjugates spontaneously self-assembled in aqueous media forming nanosized SNAs with a biodegradable polyester core and a surface of oligonucleotide chains as evidenced from dynamic and electrophoretic light scattering measurements. The nano-assemblies were in vitro evaluated for potential cytotoxicity. Furthermore, the interactions of the newly synthesized SNAs with membrane lipids were studied. The preliminary results indicate that both types of polymer-based SNAs are good candidates for potential application in gene therapy and that it is worth to be further evaluated.

Original Synthesis of a Nucleolipid for Preparation of Vesicular Spherical Nucleic Acids.

Spherical nucleic acids (SNAs)-nanostructures, consisting of a nanoparticle core densely functionalized with a shell of short oligonucleotide strands-are a rapidly emerging class of nanoparticle-based therapeutics with unique properties and specific applications as drug and nucleic acid delivery and gene regulation materials. In this contribution, we report on the preparation of hollow SNA nanoconstructs by co-assembly of an originally synthesized nucleolipid-a hybrid biomacromolecule, composed of a lipidic residue, covalently linked to a DNA oligonucleotide strand-with other lipids. The nucleolipid was synthesized via a click chemistry approach employing initiator-free, UV light-induced thiol-ene coupling of appropriately functionalized intermediates, performed in mild conditions using a custom-made UV light-emitting device. The SNA nanoconstructs were of a vesicular structure consisting of a self-closed bilayer membrane in which the nucleolipid was intercalated via its lipid-mimetic residue. They were in the lower nanometer size range, moderately negatively charged, and were found to carry thousands of oligonucleotide strands per particle, corresponding to a grafting density comparable to that of other SNA structures. The surface density of the strands on the bilayer implied that they adopted an unextended conformation. We demonstrated that preformed vesicular structures could be successfully loaded with either hydrophilic or hydrophobic dyes.

Nucleic acid-based supramolecular structures: vesicular spherical nucleic acids from a non-phospholipid nucleolipid.

Vesicular spherical nucleic acids are dynamic nucleic acid-based supramolecular structures that are held together via non-covalent bonds. They have promising applications as drug and nucleic acid delivery materials, diagnostic and imaging tools and platforms for development of various therapeutic schemes. In this contribution, we report on vesicular spherical nucleic acids, constructed from a non-phospholipid nucleolipid - an original hybrid biomacromolecule, composed of a hydrophobic residue, resembling that of the naturally occurring phospholipids, and a DNA oligonucleotide strand. The nucleolipid was synthesized by coupling of dibenzocyclooctyne-functionalized oligonucleotide and azidated 1,3-dihexadecyloxy-propane-2-ol via an azide-alkyne click reaction. In aqueous solution it spontaneously self-associated into nanosized supramolecular structures, identified as unilamellar vesicles composed of a self-closed interdigitated bilayer. Vesicular structures were also formed upon intercalation of the nucleolipid via its lipid-mimetic residue in the phospholipid bilayer membrane of liposomes prepared from readily available and FDA-approved lipids (1,2-dipalmitoyl-rac-glycero-3-phosphocholine and cholesterol). The vesicular structures are thoroughly investigated by light scattering (dynamic, static, and electrophoretic) and cryogenic TEM and the physical characteristics, in particular, number of strands per particle, grafting density, and conformation of the strands, were compared to those of reference spherical nucleic acids. Finally, the vesicular structures were shown to exhibit cellular internalization with no need of transfection agents and enhanced colloidal and nuclease stability.

Cholesterol Alters the Phase Separation in Model Membranes Containing hBest1.

Human retinal pigment epithelial (RPE) cells express the transmembrane Ca2+-dependent Cl- channel bestrophin-1 (hBest1) of the plasma membrane. Mutations in the hBest1 protein are associated with the development of distinct pathological conditions known as bestrophinopathies. The interactions between hBest1 and plasma membrane lipids (cholesterol (Chol), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and sphingomyelin (SM)) determine its lateral organization and surface dynamics, i.e., their miscibility or phase separation. Using the surface pressure/mean molecular area (π/A) isotherms, hysteresis and compressibility moduli (Cs-1) of hBest1/POPC/Chol and hBest1/SM/Chol composite Langmuir monolayers, we established that the films are in an LE (liquid-expanded) or LE-LC (liquid-condensed) state, the components are well-mixed and the Ca2+ ions have a condensing effect on the surface molecular organization. Cholesterol causes a decrease in the elasticity of both films and a decrease in the ΔGmixπ values (reduction of phase separation) of hBest1/POPC/Chol films. For the hBest1/SM/Chol monolayers, the negative values of ΔGmixπ are retained and equalized with the values of ΔGmixπ in the hBest1/POPC/Chol films. Shifts in phase separation/miscibility by cholesterol can lead to changes in the structure and localization of hBest1 in the lipid rafts and its channel functions.

Self-organization and surface properties of hBest1 in models of biological membranes.

The transmembrane Ca2+ - activated Cl- channel - human bestrophin-1 (hBest1) is expressed in retinal pigment epithelium and mutations of BEST1 gene cause ocular degenerative diseases colectivelly referred to as "bestrophinopathies". A large number of genetical, biochemical, biophysical and molecular biological studies have been performed to understand the relationship between structure and function of the hBest1 protein and its pathophysiological significance. Here, we review the current understanding of hBest1 surface organization, interactions with membrane lipids in model membranes, and its association with microdomains of cellular membranes. These highlights are significant for modulation of channel activity in cells.

A Galantamine-Curcumin Hybrid Decreases the Cytotoxicity of Amyloid-Beta Peptide on SH-SY5Y Cells.

Misfolded amyloid beta (Aß) peptides aggregate and form neurotoxic oligomers. Membrane and mitochondrial damages, calcium dysregulation, oxidative stress, and fibril deposits are among the possible mechanisms of Aß cytotoxicity. Galantamine (GAL) prevents apoptosis induced by Aß mainly through the ability to stimulate allosterically the α7 nAChRs and to regulate the calcium cytosolic concentration. Here, we examined the cytoprotective effects of two GAL derivatives, namely compounds 4b and 8, against Aß cytotoxicity on the human neuroblastoma cell line SH-SY5Y. The protective effects were tested at simultaneous administration, pre-incubation and post-incubation, with Aß. GAL and curcumin (CU) were used in the study as reference compounds. It was found that 4b protects cells in a similar mode as GAL, while compound 8 and CU potentiate the toxic effects of Aß. Allosteric stimulation of α7 nAChRs is suggested as a possible mechanism of the cytoprotectivity of 4b. These and previous findings characterize 4b as a prospective non-toxic multi-target agent against neurodegenerative disorders with inhibitory activity on acetylcholinesterase, antioxidant, and cytoprotective properties.

Condensing Effect of Cholesterol on hBest1/POPC and hBest1/SM Langmuir Monolayers.

Human bestrophin-1 protein (hBest1) is a transmembrane channel associated with the calcium-dependent transport of chloride ions in the retinal pigment epithelium as well as with the transport of glutamate and GABA in nerve cells. Interactions between hBest1, sphingomyelins, phosphatidylcholines and cholesterol are crucial for hBest1 association with cell membrane domains and its biological functions. As cholesterol plays a key role in the formation of lipid rafts, motional ordering of lipids and modeling/remodeling of the lateral membrane structure, we examined the effect of different cholesterol concentrations on the surface tension of hBest1/POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and hBest1/SM Langmuir monolayers in the presence/absence of Ca2+ ions using surface pressure measurements and Brewster angle microscopy studies. Here, we report that cholesterol: (1) has negligible condensing effect on pure hBest1 monolayers detected mainly in the presence of Ca2+ ions, and; (2) induces a condensing effect on composite hBest1/POPC and hBest1/SM monolayers. These results offer evidence for the significance of intermolecular protein-lipid interactions for the conformational dynamics of hBest1 and its biological functions as multimeric ion channel.

Miscibility of hBest1 and sphingomyelin in surface films - A prerequisite for interaction with membrane domains.

Human bestrophin-1 (hBest1) is a transmembrane Ca2+- dependent anion channel, associated with the transport of Cl-, HCO3- ions, γ-aminobutiric acid (GABA), glutamate (Glu), and regulation of retinal homeostasis. Its mutant forms cause retinal degenerative diseases, defined as Bestrophinopathies. Using both physicochemical - surface pressure/mean molecular area (π/A) isotherms, hysteresis, compressibility moduli of hBest1/sphingomyelin (SM) monolayers, Brewster angle microscopy (BAM) studies, and biological approaches - detergent membrane fractionation, Laurdan (6-dodecanoyl-N,N-dimethyl-2-naphthylamine) and immunofluorescence staining of stably transfected MDCK-hBest1 and MDCK II cells, we report: 1) Ca2+, Glu and GABA interact with binary hBest1/SM monolayers at 35 °C, resulting in changes in hBest1 surface conformation, structure, self-organization and surface dynamics. The process of mixing in hBest1/SM monolayers is spontaneous and the effect of protein on binary films was defined as "fluidizing", hindering the phase-transition of monolayer from liquid-expanded to intermediate (LE-M) state; 2) in stably transfected MDCK-hBest1 cells, bestrophin-1 was distributed between detergent resistant (DRM) and detergent-soluble membranes (DSM) - up to 30 % and 70 %, respectively; in alive cells, hBest1 was visualized in both liquid-ordered (Lo) and liquid-disordered (Ld) fractions, quantifying protein association up to 35 % and 65 % with Lo and Ld. Our results indicate that the spontaneous miscibility of hBest1 and SM is a prerequisite to diverse protein interactions with membrane domains, different structural conformations and biological functions.

Polyplex Particles Based on Comb-Like Polyethylenimine/Poly(2-ethyl-2-oxazoline) Copolymers: Relating Biological Performance with Morphology and Structure.

The present contribution is focused on feasibility of using comb-like copolymers of polyethylenimine with poly(2-ethyl-2-oxazoline) (LPEI-comb-PEtOx) with varying grafting densities and degrees of polymerization of PEI and PEtOx to deliver DNA molecules into cells. The copolymers form small and well-defined particles at elevated temperatures, which are used as platforms for binding and condensing DNA. The electrostatic interactions between particles and DNA result in formation of sub-100 nm polyplex particles of narrow size distribution and different morphology and structure. The investigated gene delivery systems exhibit transfection efficiency dependent on the copolymer chain topology, shape of the polyplex particles, and internalization pathway. Flow cytometry shows enhanced transfection efficiency of the polyplexes with elongated and ellipsoidal morphology. The preliminary biocompatibility study on a panel of human cell lines shows that pure copolymers and polyplexes thereof are practically devoid of cytotoxicity.

Effects of Ca2+, Glu and GABA on hBest1 and composite hBest1/POPC surface films.

Bestrophinopathies are ocular diseases caused by mutations in the human bestrophin-1 (hBest1) - transmembrane Ca2+-activated chloride channel protein, mainly expressed in the retinal pigment epithelium (RPE) cells. hBest1 is also an important transporter for neurotransmitters such as glutamate (Glu) and γ-aminobutyric acid (GABA) in the nervous system. Recently, a new biological role of hBest1, related to its possible involvement in the pathology of brain diseases (Alzheimer's, Parkinson's disease) has been proposed. Here, we report the effects of Ca2+, Glu and GABA on hBest1 and composite hBest1/POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) Langmuir and Langmuir-Blodgett monolayers based on surface dynamics (π/A isotherms, hysteresis and compressibility), morphology (Brewster angle microscopy, BAM) and visualization of protein molecular organization (Atomic force microscopy, AFM). Ca2+ ions and neurotransmitters Glu and GABA affect hBest1 topology at the air/water interface altering its surface activity, size, orientation and organization. In contrast, no significant changes were detected on π/A isotherms and hysteresis of the composite hBest1/POPC films but their effects on structure, aggregation state and orientation hBest1 established by BAM and AFM differentiate. We found that the binary films of hBest1 and POPC are phase separated at the air/water interface, suggesting stronger lipid-lipid and protein-protein interactions than lipid-protein interactions that can significantly alter the molecular organization and activity of hBest1 in cell membranes. Our data shed light on structure, surface behavior and organization of hBest1 that define relationship structure-functional activity of hBest1 as transport channel.

Effects of Ca2+ ions on bestrophin-1 surface films.

Human bestrophin-1 (hBest1) is a transmembrane calcium-activated chloride channel protein - member of the bestrophin family of anion channels, predominantly expressed in the membrane of retinal pigment epithelium (RPE) cells. Mutations in the protein cause ocular diseases, named Bestrophinopathies. Here, we present the first Fourier transform infrared (FTIR) study of the secondary structure elements of hBest1, π/A isotherms and hysteresis, Brewster angle microscopy (BAM) and atomic force microscopy (AFM) visualization of the aggregation state of protein molecules dispersed as Langmuir and Langmuir-Blodgett films. The secondary structure of hBest1 consists predominantly of 310-helices (27.2%), α-helixes (16.3%), ß-turns and loops (32.2%). AFM images of hBest1 suggest approximate lateral dimensions of 100×160Å and 75Å height. Binding of calcium ions (Ca2+) induces conformational changes in the protein secondary structure leading to assembly of protein molecules and changes in molecular and macro-organization of hBest1 in monolayers. These data provide basic information needed in pursuit of molecular mechanisms underlying retinal and other pathologies linked to this protein.

Transepithelial resistance in human bestrophin-1 stably transfected Madin-Darby canine kidney cells.

Bestrophin-1 (Best1) is a transmembrane protein, found in the basolateral plasma membrane of retinal pigmented epithelial cells. The exact structure and functions of Best1 protein are still unclear. The protein is thought to be a regulator of ion channels, or an ion channel itself: it was shown to be permeable for chloride, thiocyanate, bicarbonate, glutamate and γ-aminobutyric acid (GABA). Mutations in the gene for Best1 are leading to best vitelliform macular dystrophy (BVMD) and are found in several other types of maculopathy. In order to obtain additional information about Best1 protein, we determined cell polarization of a stably transfected Madin-Darby canine kidney cell line II (MDCK II) cell line, expressing human Best1. We measured the transepithelial resistance of transfected and non-transfected MDCK cells by voltmeter EVOM, over 10 days at 24 hour intervals. The first few days (first-fourth day) both cell lines showed the same or similar values ​​of transmembrane resistance. As expected, on the fifth day the non-transfected cells showed maximum value of epithelial resistance, corresponding to the forming of monolayer. The transfected cells showed maximum value of transepithelial resistance on the ninth day of their cultivation. Phalloidin staining of actin demonstrated the difference in actin arrangements between transfected and non-transfected cells due to Best1. As a consequence of actin rearrangement, Best1 strongly affects the transepithelial resistance of polarizing stably transfected MDCK cells. Our results suggest that Best1 protein has an effect on transepithelial resistance and actin rearrangements of polarized stably transfected MDCK cells.

Changes in the functional characteristics of tumor and normal cells after treatment with extracts of white dead-nettle.

Lamium album L. is a perennial herb widely used in folk medicine. It possesses a wide spectrum of therapeutic activities (anti-inflammatory, astringent, antiseptic, antibiotic, antispasmodic, antioxidant and anti-proliferative). Preservation of medicinal plant could be done by in vitro propagation to avoid depletion from their natural habitat. It is important to know whether extracts from L. album plants grown in vitro possess similar properties as extracts from plants grown in vivo. For these reasons, it is important to examine changes in the composition of secondary metabolites during in vitro cultivation of the plant and how they affect the biological activity. We used A549 human cancer cell line and normal kidney epithelial cells MDCKII (Madin-Darby canine kidney cells II) as controls in assessing the anti-cancer effect of plant extracts. To elucidate changes in some key functional characteristics, adhesion test, MTT (3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyl tetrazolium bromide), transepithelial resistance (TER), immunofluorescence staining and trypan blue exclusion test were performed. Methanol and chloroform extracts of in vivo and in vitro propagated plants affected differently cancerous and non-cancerous cells. The most pronounced differences were observed in the morphological analysis and in the cell adhesive properties. We also detected suppressed epithelial transmembrane electrical resistance of MDCK II cells, by treatment with plant extracts, compared to non-treated MDCK II cells. A549 cells did not polarize under the same conditions. Altered organization of actin filaments in both cell types were noticed suggesting that extracts from L. album L. change TER and actin filaments, and somehow may block cell mechanisms, leading to the polarization of MDCK II cells.

Effects of vipoxin and its components on HepG2 cells.

Snake venom Phospholipases A2 (svPLA2) are among the main toxic venom components with a great impact on different tissues and organs based on their catalytic specificity and a variety of pharmacological effects, whose mechanism is still under debate. The main toxic component, isolated from the venom of Vipera ammodytes meridionalis, is the heterodimeric postsynaptic ionic complex vipoxin, composed of a basic and toxic PLA2 enzyme subunit (GIIA secreted PLA2) and an acidic, enzymatically inactive and nontoxic subunit - vipoxin acidic component (VAC). This study demonstrates for the first time that vipoxin and its individual subunits affect integrity and viability of HepG2 cells displaying differences in their pharmacological activities. Under the experimental conditions, the individual PLA2 subunit induces cytotoxicity, cytoskeletal rearrangements and triggers early apoptosis in a concentration-dependent manner related to its enzymatic activity. Vipoxin and VAC do not affect cell viability but manifest high degree of genotoxicity, whereas DNA damage induced by PLA2 subunit could be defined as moderate and not associated with its catalytic activity. Our results suggest that the interactions between vipoxin subunits play an important role in HepG2 cell response and most likely affect the observed distinction between cyto- and genotoxicity.

Interaction of Bestrophin-1 with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in surface films.

Human bestrophin-1 (hBest1) is a transmembrane channel protein, predominantly expressed in the membrane of retinal pigment epithelium (RPE) cells. Although it is clear that hBest1's interactions with lipids are crucial for its function such studies were not performed as the protein was not purified. Here we describe an effective purification of hBest1 from Madin-Darby Canine Kidney (MDCK) cells via simple gel-filtration and affinity chromatographic steps, which makes possible to probe the protein interplay with lipids. The interaction of the purified hBest1 with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was studied in Langmuir monolayers. The surface pressure (π)-area (A) isotherms and compression/expansion isocycles of POPC monolayer were recorded in absence and presence of hBest1 in the subphase. The π(A) isotherms were analyzed in terms of surface compressional modulus and via two-dimensional virial equation of state. The dilatational rheological properties of the surface films and their surface potential were also measured. The morphology of the films was observed by Brewster angle microscopy. The inclusion of the protein in the film subphase does not lead to in-depth penetration of hBest1 but interaction takes place in the headgroup region of the monolayer. The hBest1/POPC interaction resulted in formation of more condensed films, which rheological properties and lateral structure differed significantly from the pure POPC monolayers. Our study sheds light on the still unclear question how hBest1 gets in touch with biomembrane phospholipids of eukaryotic cells that might be of key importance for the proper structure and function of RPE biomembranes.

Interactions of pharmacologically active snake venom sPLA2 with different cell lines.

Secreted Phospholipases A2 (sPLA2s) represent a large family of structurally related enzymes, which target different tissues and organs and induce numerous pharmacological effects based on their catalytic specificity - hydrolysis of the sn-2 ester bond of glycerophospholipids. The neurotoxin vipoxin, isolated from the venom of Vipera ammodytes meriodionalis, is a heterodimeric postsynaptic ionic complex composed of two protein subunits - a basic and toxic His48 sPLA2 enzyme and an acidic, enzymatically inactive and non-toxic component. In this paper, for the first time, we demonstrate that vipoxin sPLA2 enzyme affects cell integrity and viability of four cell types and causes different cell responses. The most dramatic local tissue effects were observed with RPE-1 (retinal pigment epithelial) cells followed by A549 (adenocarcinomic human alveolar epithelial) cells and MDCK (Madin-Darby Canine Kidney epithelial) cells. Products of the enzymatic reaction, lysophospholipids and unsaturated free fatty acids, act as lipid mediators that can induce membrane damaging or can stimulate cell proliferation. Our preliminary results on the cytotoxic effect of vipoxin sPLA2 on A549 cells are promising in searching of its eventual anticancer potential.