Urszula Bazylińska

Novel approach to long sustained multilayer nanocapsules: influence of surfactant head groups and polyelectrolyte layer number on the release of hydrophobic compounds

Nanoemulsion-templated long sustained polyelectrolyte (PE) nanocapsules (average size < 200 nm) loaded with Oil Red O and two cyanine-type photosensitizers: IR-786 and IR-780 were successfully fabricated by using the layer-by-layer (LbL) technique. All nanoproducts were subjected to in vitro release characteristics and analysis of selected control parameters, i.e., type of surfactant head group, characteristic release time and surfactant–polyelectrolyte interactions. Their properties were characterized by means of dynamic light scattering (DLS) compared with scanning electron microscopy (SEM) and atomic force microscopy (AFM). In our studies for construction of oil-in-water nanoemulsion templates we selected three cationic surfactants with different nature of hydrophilic head groups, i.e., double-headed (or so-called dicephalic-type) N,N-bis[3,3′(trimethylammonio)propyl]dodecanamide dimethylsulfate (C12(TAPAMS)2), bulky saccharide-derived 2-(dodecyldimethylammonio)ethylglucoheptonamide bromide (D2GHA-12) and a classic dodecyltrimethylammonium bromide (DTABr) for comparison. The polyelectrolytes were the following: polyanion of poly(sodium 4-styrenesulfonate) (PSS) and polycation of poly(diallyldimethylammonium chloride) (PDADMAC). The in vitro release profile features, studied spectrophotometrically, were interpreted in the framework of diffusion-controlled processes and stability of the first interfacial PE–surfactant complex. Accordingly, the multicharge and bulky structure of the surfactant are found to be the most desirable factors for fabrication of long sustained and stable nanocapsules encapsulating a hydrophobic active substance.

Biocompatible long-sustained release oil-core polyelectrolyte nanocarriers: From controlling physical state and stability to biological impact

It has been generally expected that the most applicable drug delivery system (DDS) should be biodegradable, biocompatible and with incidental adverse effects. Among many micellar aggregates and their mediated polymeric systems, polyelectrolyte oil-core nanocarriers have been found to successfully encapsulate hydrophobic drugs in order to target cells and avoid drug degradation and toxicity as well as to improve drug efficacy, its stability, and better intracellular penetration. This paper reviews recent developments in the formation of polyelectrolyte oil-core nanocarriers by subsequent multilayer adsorption at micellar structures, their imaging, physical state and stability, drug encapsulation and applications, in vitro release profiles and in vitro biological evaluation (cellular uptake and internalization, biocompatibility). We summarize the recent results concerning polyelectrolyte/surfactant interactions at interfaces, fundamental to understand the mechanisms of formation of stable polyelectrolyte layered structures on liquid cores. The fabrication of emulsion droplets stabilized by synergetic surfactant/polyelectrolyte complexes, properties, and potential applications of each type of polyelectrolyte oil-core nanocarriers, including stealth nanocapsules with pegylated shell, are discussed and evaluated.

Nanoemulsion-templated multilayer nanocapsules for cyanine-type photosensitizer delivery to human breast carcinoma cells.

There is great clinical interest in developing novel nanocarriers for hydrophobic cyanine dyes used as photosensitizing agents in photodynamic therapy (PDT). In the present study we have employed nanoemulsion-templated oil-core multilayer nanocapsules as robust nanocarriers for a cyanine-type photosensitizer IR-786. These nanoproducts were fabricated via layer-by-layer (LbL) adsorption of oppositely charged polyelectrolytes (PEs), i.e., anionic PSS and cationic PDADMAC on nanoemulsion liquid cores created by dicephalic or bulky saccharide-derived cationic surfactants. All nanocapsules, with different thicknesses of the PE shell and average size <200 nm (measured by DLS) demonstrated good capacity for IR-786 encapsulation. The nanocarriers were visualized by SEM and AFM and their photo-induced anticancer effect and cellular internalization in human breast carcinoma MCF-7/WT cells were determined. Biological response of the cell culture, expressed as dark and photocytotoxicity as well as fluorescence of drug molecules loaded in the multilayer vehicles, analyzed by the FACS and CLSM techniques, have indicated that the delivered IR-786 did not aggregate inside the cells and could, therefore, act as an effective third-generation photosensitizing agent. In vitro biological experiments demonstrated that the properties of studied nanostructures depended upon the PE type and the envelope thickness as well as on the surfactant architecture in the nanoemulsion-based templates employed for the nanocapsule fabrication. Similarity of results obtained for stored (three weeks in the dark at room temperature) and freshly-prepared nanocapsules, attests to viability of this stable, promising drug delivery system for poorly water-soluble cyanines useful in PDT.

Polymeric micelles for enhanced Photofrin II® delivery, cytotoxicity and pro-apoptotic activity in human breast and ovarian cancer cells

BACKGROUND Searching for photodynamic therapy (PDT) - effective nanocarriers which enable a photosensitizer to be selectively delivered to tumor cells with enhanced bioavailability and diminished dark cytotoxicity is of current interest. The main objective of this study is to evaluate newly designed mixed polymeric micelles based on Pluronics P123 and F127 for the improved delivery of Photofrin II(®) (Ph II(®)) to circumvent unfavorable effects overcoming multidrug resistance (MDR) in tumor cells - in breast MCF-7/WT (caspase-3 deficient) and ovarian SKOV-3 (resistant to chemotherapy). METHODS Ph II(®)-loaded micelles were obtained and analyzed for size and morphology, solubilization efficiency, physical stability and in vitro drug release. Intracellular uptake, reactive oxygen species (ROS) generation, mitochondrial oxidoreductive potential and proapoptotic activity (TUNEL assay) studies were evaluated in the examined cancer cells. The preliminary biocompatibility characteristics of all nanocarriers was determined by assessment of their hemolytic activity in human erythrocytes and dark toxicity in cancer cells. RESULTS Dynamic light scattering (DLS) and atomic force microscopy (AFM) confirmed that almost monodisperse, sphere-shaped and nanosized (DH<20 nm) carriers were developed. Biological studies after photodynamic reaction (PDR) with encapsulated Ph II(®) revealed increased ROS level, malondialdehyde (MDA) concentration and protein damage in SKOV-3 and MCF-7/WT cells in comparison to treatment with free Ph II(®). Numerous apoptotic cells were detected after nano-therapy in both cell lines, with observed significant morphological disorders in ovarian cancer cells. In the case of encapsulated Ph II(®) only negligible disruption of human erythrocytes and cancer cells was observed. CONCLUSIONS The obtained biocompatible long-lasting nanocarriers significantly enhance the Photofrin II(®) photodynamic effect and apoptosis in both SKOV-3 and MCF-7/WT cell lines.

Core/shell quantum dots encapsulated in biocompatible oil-core nanocarriers as two-photon fluorescent markers for bioimaging.

Highly fluorescent quantum dots (QDs)-loaded nanocapsules, intended for fluorescent cell imaging, were prepared via an emulsification/solvent-evaporation method. CdSe/ZnS core/shell quantum dots were applied as cargo; Poloxamer 403 as the polymer component; Cremophor EL as the nonionic surfactant; and mineral oil, oleic acid, or silicone oil were applied as the oil phases. Transmission electron microscopy, atomic force microscopy, dynamic light scattering, and zeta potential measurements were used to characterize the novel QDs-labeled nanoparticles by particle size, distribution, and morphology, as well as by ζ-potential and physical stability. The fabricated long-lasting nanocapsules exhibit good luminescence properties upon both one-photon and two-photon excitation. The potential of the encapsulated QDs for fluorescent imaging was evaluated in cytotoxicity studies as well as in imaging of intracellular localization, accumulation, and distribution of QDs delivered to well-characterized human cancer cell lines--doxorubicin-sensitive breast (MCF-7/WT) and alveolar basal epithelial (A549)--as well as on normal human umbilical vein endothelial (HUVEC) cells, as investigated by confocal laser scanning microscopy (CLSM). The colloidal CdSe/ZnS-loaded nanocapsules are shown to exhibit strong two-photon-induced luminescence upon excitation in the NIR optical transmission window spectral range, making them ideal markers for bioimaging application. The total two-photon cross section of a single nanocapsule was determined to be about 4.1 × 10(6) GM at 800 nm.

New diamidequat-type surfactants in fabrication of long-sustained theranostic nanocapsules: Colloidal stability, drug delivery and bioimaging

We report a new theranostic nanoformulation to transport both chemotherapeutic and imaging agents for successfully exterminating cancer cells. This strategy is based on encapsulation of colchicine (cytostatic drug) and coumarin-6 (fluorescent biomarker) in oil-core nanocarriers stabilized by diamidequat-type surfactants - N,N-dimethyl-N,N-bis[2-(N-alkylcarbamoyl) ethyl]ammonium methylsulfates (2xCnA-MS, n=8,10,12), and fabricated by the nanoprecipitation technique. The surfactants were synthesized using a technologically viable methodology and characterized. The potential of the encapsulated theranostic cargoes was evaluated in cytotoxicity studies as well as in imaging of intracellular localization, accumulation and distribution of cargoes delivered to well characterized human cancer cell lines - doxorubicin-sensitive breast (MCF-7/WT), alveolar basal epithelial (A549) and skin melanoma (MEWO) - performed by confocal laser scanning microscopy (CLSM). Backscattered profiles obtained by the turbidimetric technique were applied to evaluate physical stability of the obtained nanosystems. DLS measurements confirmed the particle diameter to be below 200nm, while AFM - its morphology and shape. Doppler electrophoresis provided a highly positive ζ-potential. UV-vis was applied to determine the encapsulation efficiencies (ca. 90%), and release profiles. The study demonstrates that the soft cationic diamidequat-type surfactants are suitable for the stabilization of theranostic nanodispersions, and they can constitute a new functional class of stabilizers of nanoparticles and have a progressive impact onto development of formulations. Furthermore, our results demonstrate excellent biocompatibility of the studied long-sustained monodisperse oil-core nanocapsules, stabilized by 2xCnA-MS, which makes them promising for cell imaging.

Microfluidic platform for photodynamic therapy cytotoxicity analysis of nanoencapsulated indocyanine-type photosensitizers

The application of nanotechnology is important to improve research and development of alternative anticancer therapies. In order to accelerate research related to cancer diagnosis and to improve the effectiveness of cancer treatment, various nanomaterials are being tested. The main objective of this work was basic research focused on examination of the mechanism and effectiveness of the introduction of nanoencapsulated photosensitizers to human carcinoma (A549) and normal cells (MRC-5). Newly encapsulated hydrophobic indocyanine-type photosensitizer (i.e., IR-780) was subjected to in vitro studies to determine its release characteristics on a molecular level. The photosensitizers were delivered to carcinoma and normal cells cultured under model conditions using multiwell plates and with the use of the specially designed hybrid (poly(dimethylsiloxane) (PDMS)/glass) microfluidic system. The specific geometry of our microsystem allows for the examination of intercellular interactions between cells cultured in the microchambers connected with microchannels of precisely defined length. Our microsystem allows investigating various therapeutic procedures (e.g., photodynamic therapy) on monoculture, coculture, and mixed culture, simultaneously, which is very difficult to perform using standard multiwell plates. In addition, we tested the cellular internalization of nanoparticles (differing in size, surface properties) in carcinoma and normal lung cells. We proved that cellular uptake of nanocapsules loaded with cyanine IR-780 in carcinoma cells was more significant than in normal cells. We demonstrated non cytotoxic effect of newly synthesized nanocapsules built with polyelectrolytes (PEs) of opposite surface charges: polyanion-polysodium-4-styrenesulphonate and polycation-poly(diallyldimethyl-ammonium) chloride loaded with cyanine IR-780 on human lung carcinoma and normal cell lines. However, the differences observed in the photocytotoxic effect between two types of tested nanocapsules can result from the type of last PE layer and their different surface charge.

Evaluation of nanoencapsulated verteporfin's cytotoxicity using a microfluidic system.

A new-generation of nanoencapsulated photosensitizers could be a good solution to perform effective photodynamic therapy (PDT). In this paper, we present physicochemical characterization and cellular investigation of newly prepared long-sustained release oil-core polyelectrolyte nanocarriers loaded with verteporfin (nano VP) in relation to free VP. For this purpose, a macroscale multiwell plates and multifunctional microfluidic system (for three types of cell cultures: monoculture, coculture and mixed culture) were used. A physical analysis of nano VP showed its high stability, monodispersity with unimodal shape and highly positive charge, what made them good candidates for cancer treatment. Biological properties (cellular internalization and uptake as well as cytotoxicity) of nano and free VP were evaluated using both carcinoma (A549) and normal (MRC-5) human lung cells. It was investigated that verteporfin was accumulated in cancer cells preferentially. Low cytotoxicity of the tested photosensitizer was observed in both macro, and microscale. However, in experiments performed in the microsystem, nano VP allowed the reduction of cytotoxic effect, especially in relation to the normal cells. It could result from the specific environment of cell growth in the microsystem which can quite closely mimic the in vivo conditions. Our results suggest that the presented microsystem could be a very useful microtool for testing of new generation of photosensitizers in various configurations of cell cultures, which are difficult to perform in the macroscale. Moreover, the prepared nano VP could be successfully used for further research i.e. evaluation of PDT procedures.

In vitro studies of serum albumin interaction with poly(D,L-lactide) nanospheres loaded by hydrophobic cargo.

The various polymer-based nanocarriers are very attractive for in vitro and in vivo bioapplications. A new type of a promising drug delivery systems for cancer tissues-poly(D,L-lactide) nanospheres stabilized with Cremophor EL and loaded with hydrophobic cyanines (IR-780 or ZnPc) or curcumin (CUR) were fabricated by the nanoprecipitation method. The Cremophor EL/PLA/water nanospheres demonstrated regular shape, low polydispersity (PdI<0.3) and high entrapment efficiency of selected cargo (over 90%). The size of those nanoconstructs below 130 nm are in the desired nanocarriers size range for tumor delivery. Low level of in vitro drug release from loaded nanospheres after long-time storage indicates their good stability. The half-life of nanocarriers in the circulation, and their biodistribution after parenteral administration are associated with the ability of plasma proteins adsorption. For these reasons the affinity of obtained nanospheres for albumin as a major plasma protein was in vitro investigated. The binding of nanocarrier containing cyanine IR-780 with albumin immobilized in the wells of polystyrene plate occurred with lower efficiency than analogs loaded with ZnPc or CUR. Similar relationships were observed after UV-vis spectra analysis of nanospheres in the presence of albumin at various protein concentrations.

Optical, colloidal and biological properties of up-converting nanoparticles embedded in polyester nanocarriers

We have investigated the change in optical properties and biocompatibility of up-converting NaYF4 nanoparticles (NPs) upon encapsulation inside the polyester nanocarriers (NCs) stabilized by Crempophor RH40 (CRH40), poly(D,L-lactide) (PLA), Pluronic P123 (P123). NaYF4:Er3+,Yb3+ NPs showed intense green and red emission, and upon encapsulation the increase of red band in respect to green one was observed, with no luminescence lifetime shortening. Obtained NCs showed prolonged colloidal stability and protective effect of the polymer shell simultaneously preserving the high emission efficiency of nanoparticles embedded within the silicon oil (SO) core. Based on emission spectra, kinetic measurements and cytotoxicity studies upon human malignant melanoma Me45 cell line we have shown the advantages of using polyester NCs as containers for the up-converting NPs. Due to the possibility of co-encapsulation of photosensitizers the obtained nanocarriers showed potential for application in theranostics.