Investigating 3R In Vivo Approaches for Bio-Distribution and Efficacy Evaluation of Nucleic Acid Nanocarriers: Studies on Peptide-Mimicking Ionizable Lipid.

Formulations based on ionizable amino-lipids have been put into focus as nucleic acid delivery systems. Recently, the in vitro efficacy of the lipid formulation OH4:DOPE has been explored. However, in vitro performance of nanomedicines cannot correctly predict in vivo efficacy, thereby considerably limiting pre-clinical translation. This is further exacerbated by limited access to mammalian models. The present work proposes to close this gap by investigating in vivo nucleic acid delivery within simpler models, but which still offers physiologically complex environments and also adheres to the 3R guidelines (replace/reduce/refine) to improve animal experiments. The efficacy of OH4:DOPE as a delivery system for nucleic acids is demonstrated using in vivo approaches. It is shown that the formulation is able to transfect complex tissues using the chicken chorioallantoic membrane model. The efficacy of DNA and mRNA lipoplexes is tested extensively in the zebra fish (Danio rerio) embryo which allows the screening of biodistribution and transfection efficiency. Effective transfection of blood vessel endothelial cells is seen, especially in the endocardium. Both model systems allow an efficacy screening according to the 3R guidelines bypassing the in vitro-in vivo gap. Pilot studies in mice are performed to correlate the efficacy of in vivo transfection.

Liposome-Encapsulated Bioactive Guttiferone E Exhibits Anti-Inflammatory Effect in Lipopolysaccharide-Stimulated MH-S Macrophages and Cytotoxicity against Human Cancer Cells.

Background: Guttiferone E is a naturally occurring polyisoprenylated benzophenone exhibiting a wide range of remarkable biological activities. But its therapeutic application is still limited due to its poor water solubility. This study is aimed at preparing guttiferone E-loaded liposomes and assessing their in vitro cytotoxicity and anti-inflammatory effect. Methods: Liposomes containing guttiferone E were prepared by the thin film hydration method, and the physicochemical characteristics were determined using dynamic light scattering, laser Doppler velocimetry, and atomic force microscopy. The cytotoxicity was assessed by the MTT assay. The fluorometric cyclooxygenase (COX) activity assay kit was used to assess the COX activity while the nitric oxide production was evaluated by the Griess reagent method. Results: The liposomes with a mean size of 183.33 ± 17.28 nm were obtained with an entrapment efficiency of 63.86%. Guttiferone E-loaded liposomes successfully decreased the viability of cancer cells. The overall IC50 values varied between 5.46 µg/mL and 22.25 µg/mL. Compared to the untreated control, guttiferone E-loaded liposomes significantly reduced the nitric oxide production and the activity of COX in a concentration-dependent manner. Conclusion: This study indicates that liposomes can be an alternative to overcome the water insolubility issue of the bioactive guttiferone E.

In situ intravenous photodynamic therapy for the systemic eradication of blood stream infections.

Photodynamic therapy is one of the most promising non-invasive strategies employed for the treatment of several kinds of bacterial infections. Though the vast majority of clinically approved photosensitisers are administered intravenously, most of the in vitro experiments are performed under static conditions which do not represent the physiological environment of the venous bloodstream. To address this issue, a dynamic circulation model was developed to facilitate in situ antibacterial photodynamic therapy under flow conditions to mimic blood stream infections.

Transfection Studies with Colloidal Systems Containing Highly Purified Bipolar Tetraether Lipids from Sulfolobus acidocaldarius.

Lipid vectors are commonly used to facilitate the transfer of nucleic acids into mammalian cells. In this study, two fractions of tetraether lipids from the archaea Sulfolobus acidocaldarius were extracted and purified using different methods. The purified lipid fractions polar lipid fraction E (PLFE) and hydrolysed glycerol-dialkyl-nonitol tetraether (hGDNT) differ in their structures, charge, size, and miscibility from conventional lipids. Liposomes were prepared by mixing tetraether lipids with cholesterol (CH) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) resulting in stable vectors for gene delivery. Lipoplexes were prepared by complexation of liposomes with a luciferase expressing plasmid (pCMV-luc) at certain nitrogen-to-phosphorus (N/P) ratios and optimised for the transient transfection of ovarian adenocarcinoma cells (SK-OV-3). Complexation efficacy was investigated by gel-red fluorescence assay. Biophysical properties, like size, surface charge, and morphology, were investigated by differential light scattering (DLS), atomic force microscopy (AFM), and scanning electron microscopy (Cryo-SEM), respectively, revealing structural differences between liposomes and lipoplexes. A range of stable transfecting agents containing tetraether lipids were obtained by incorporating 5 mol% of tetraether lipids. Lipoplexes showed a decrease in free gel-red with increasing N/P ratios indicating efficient incorporation of plasmid DNA (pDNA) and remarkable stability. Transfection experiments of the lipoplexes revealed successful and superior transfection of SK-OV-3 cell line compared to the commercially available DOTAP and branched polyethyleneimine (25 kDa bPEI).

Liposome-polyethylenimine complexes (DPPC-PEI lipopolyplexes) for therapeutic siRNA delivery in vivo.

Therapeutic applications of RNA interference (RNAi) require efficient siRNA delivery strategies in vivo. Combining lipid-based carriers with polymeric nanoparticles offers the favorable properties of both systems. This is the first study to explore polyethylenimine-based lipopolyplexes comprising a low-molecular weight PEI and the phospholipid DPPC for therapeutic siRNA use. Lipopolyplex structures are analyzed by electron microscopy. Biological efficacies are demonstrated in vitro by cellular uptake, knockdown of the target oncogene survivin, and concomitant cell growth inhibition. Upon systemic administration in tumor-bearing mice, here performed by intraperitoneal (i.p.) injection, radioactive biodistribution assays show lipopolyplex-mediated delivery of intact siRNAs. Absence of blood serum parameter alterations, erythrocyte aggregation or immunostimulation, and the observation of animal well-being and stable body weight confirm biocompatibility. Exploring therapeutic efficacies in a preclinical model, a considerable inhibition of prostate carcinoma xenograft growth is achieved, paralleled by an ~65% survivin knockdown in the tumors. We, thus, demonstrate that PEI-based lipopolyplexes represent an efficient platform for therapeutic use of small RNAs.

In Vitro Photoselective Gene Transfection of Hepatocellular Carcinoma Cells with Hypericin Lipopolyplexes.

The lipopolyplex, a multicomponent nonviral gene carrier, generally demonstrates superior colloidal stability, reduced cytotoxicity, and high transfection efficiency. In this study, a new concept, photochemical reaction-induced transfection, using photosensitizer (PS)-loaded lipopolyplexes was applied, which led to enhanced transfection and cytotoxic effects by photoexcitation of the photosensitizer. Hypericin, a hydrophobic photosensitizer, was encapsulated in the lipid bilayer of liposomes. The preformed nanosized hypericin liposomes enclosed the linear polyethylenimine (lPEI)/pDNA polyplexes, resulting in the formation of hypericin lipopolyplexes (Hy-LPP). The diameters of Hy-LPP containing 50 nM hypericin and 0.25 µg of pDNA were 185.6 ± 7.74 nm and 230.2 ± 4.60 nm, respectively, measured by dynamic light scattering (DLS) and atomic force microscopy (AFM). Gel electrophoresis confirmed the encapsulation of hypericin and pDNA in lipopolyplexes. Furthermore, in vitro irradiation of intracellular Hy-LPP at radiant exposures of 200, 600, and 1000 mJ/cm2 was evaluated. It demonstrated 60- to 75-fold higher in vitro luciferase expression than that in nonirradiated cells. The lactate dehydrogenase (LDH) assay supported that reduced transfection was a consequence of photocytotoxicity. The developed photosensitizer-loaded lipopolyplexes improved the transfection efficiency of an exogenous gene or induced photocytotoxicity; however, the frontier lies in the applied photochemical dose. The light-triggered photoexcitation of intracellular hypericin resulted in the generation of reactive oxygen species (ROS), leading to photoselective transfection in HepG2 cells. It was concluded that the two codelivered therapeutics resulted in enhanced transfection and a photodynamic effect by tuning the applied photochemical dose.

Hair follicle targeting with curcumin nanocrystals: Influence of the formulation properties on the penetration efficacy.

Nanocrystals are a universal formulation approach for improved drug delivery of poorly water-soluble drug substances. Besides oral application, also topical application of the nanocrystals is feasible, because the increased kinetic solubility of the nanocrystals results in an increased concentration gradient, thus fostering passive, dermal penetration. Nanocrystals are also promising for targeting drug substances into the hair follicle. After penetration into the hair follicle, the nanocrystals could form a depot from which the active is released into the hair follicle. Thus, leading to a long-lasting and very efficient dermal drug delivery. The efficacy of nanocrystals to penetrate the hair follicles and the influence of the vehicle in which the nanocrystals are suspended was not yet investigated. Therefore, in this study curcumin nanocrystals with a size of about 300 nm were produced and incorporated into gels with different properties. The efficacy to penetrate the hair follicles, as well as the passive, dermal penetration, was assessed on the ex-vivo pig ear model. Nanocrystals were efficiently taken up by the hair follicles and reached the lower part of the infundibulum. This region is optimal for efficient drug delivery because the barrier of the lower infundibulum is not fully developed and thus more permeable, which results in a less hindered passive diffusion of drug substances. The penetration efficacy of the nanocrystals into the hair follicles was not affected by the different types of vehicles, which represented either oleogels or hydrogels that varied in viscosity as well as in the type and the concentration of the gelling agent. All gels possessed a shear-thinning flow behavior and it is hypothesized that all gels fluidized during the skin massage, whereby leading to similarly low viscosities than the aqueous nanosuspension and thus to similar penetration results. The passive, dermal penetration of curcumin was different for the different gels and the main driving parameter leading to good passive diffusion was caused by good skin hydrating properties of the vehicle. The best passive penetration was achieved from hydrogels that contained a humectant. However, the addition of the humectant reduced the efficacy of the nanocrystals to penetrate the hair follicle. Data so far, therefore, suggest that hair follicle targeting with nanocrystals that are suspended in water or simple, shear-thinning gels is highly effective. However, the addition of other excipients, e.g. humectants, to these vehicles might cause changes in the penetration profiles. More research in this regard is needed to understand these observations in more detail.

The chorioallantoic membrane as a bio-barrier model for the evaluation of nanoscale drug delivery systems for tumour therapy.

In 2010, the European Parliament and the European Union adopted a directive on the protection of animals used for scientific purposes. The directive aims to protect animals in scientific research, with the final goal of complete replacement of procedures on live animals for scientific and educational purposes as soon as it is scientifically viable. Furthermore, the directive announces the implementation of the 3Rs principle: "When choosing methods, the principles of replacement, reduction and refinement should be implemented through a strict hierarchy of the requirement to use alternative methods." The visibility, accessibility, and the rapid growth of the chorioallantoic membrane (CAM) offers a clear advantage for various manipulations and for the simulation of different Bio-Barriers according to the 3R principle. The extensive vascularisation on the CAM provides an excellent substrate for the cultivation of tumour cells or tumour xenografts which could be used for the therapeutic evaluation of nanoscale drug delivery systems. The tumour can be targeted either by topical application, intratumoural injection or i.v. injection. Different application sites and biological barriers can be examined within a single model.

Lipoparticles for Synergistic Chemo-Photodynamic Therapy to Ovarian Carcinoma Cells: In vitro and in vivo Assessments.

PURPOSE: Lipoparticles are the core-shell type lipid-polymer hybrid systems comprising polymeric nanoparticle core enveloped by single or multiple pegylated lipid layers (shell), thereby melding the biomimetic properties of long-circulating vesicles as well as the mechanical advantages of the nanoparticles. The present study was aimed at the development of such an integrated system, combining the photodynamic and chemotherapeutic approaches for the treatment of multidrug-resistant cancers. METHODS: For this rationale, two different sized Pirarubicin (THP) loaded poly lactic-co-glycolic acid (PLGA) nanoparticles were prepared by emulsion solvent evaporation technique, whereas liposomes containing Temoporfin (mTHPC) were prepared by lipid film hydration method. Physicochemical and morphological characterizations were done using dynamic light scattering, laser doppler anemometry, atomic force microscopy, and transmission electron microscopy. The quantitative assessment of cell damage was determined using MTT and reactive oxygen species (ROS) assay. The biocompatibility of the nanoformulations was evaluated with serum stability testing, haemocompatibility as well as acute in vivo toxicity using female albino (BALB/c) mice. RESULTS AND CONCLUSION: The mean hydrodynamic diameter of the formulations was found between 108.80 ± 2.10 to 405.70 ± 10.00 nm with the zeta (ζ) potential ranging from -12.70 ± 1.20 to 5.90 ± 1.10 mV. Based on the physicochemical evaluations, the selected THP nanoparticles were coated with mTHPC liposomes to produce lipid-coated nanoparticles (LCNPs). A significant (p< 0.001) cytotoxicity synergism was evident in LCNPs when irradiated at 652 nm, using an LED device. No incidence of genotoxicity was observed as seen with the comet assay. The LCNPs decreased the generalized in vivo toxicity as compared to the free drugs and was evident from the serum biochemical profile, visceral body index, liver function tests as well as renal function tests. The histopathological examinations of the vital organs revealed no significant evidence of toxicity suggesting the safety and efficacy of our lipid-polymer hybrid system.

Comparison of Tanaka lipid mixture with natural surfactant Alveofact to study nanoparticle interactions on Langmuir film balance.

Upon inhalation, nanoparticles enter the lungs where the pulmonary surfactant forms the first point of contact and plays a pivotal role for the subsequent absorption into the body. This can lead to interactions that alter the biophysical function of the surfactant monolayer. Therefore, a reliable prediction of the interaction is desired. In this study, we compared the behaviour of an artificial surfactant model with that of a natural surfactant upon exposure to chitosan nanoparticles. To simulate the physiology of the lungs, the surfactant monolayers were placed at an air/aqueous interface of a Langmuir film balance. Based on the data obtained from the experiments, the chitosan nanoparticles first integrated into the monolayer of the natural surfactant and then interact strongly with its compounds thereby moving out of the monolayer. The topographic changes in the monolayer were determined by atomic force microscopy analysis. Using this technique, the nanoparticle localisation on the monolayer could be studied. No visible interaction was observed with the artificial surfactant from surface pressure-time isotherms and atomic force microscopy analysis. Incomplete miscibility lead to instability of the artificial surfactant which left behind a DPPC rich monolayer after nanoparticle interaction. It was not stable enough to see a possible interaction (i.e. change in surface pressure) with the nanoparticles directly. These results should help understand the interactions of lipids among themselves and with the nanoparticles. Furthermore, it should help generate an efficient artificial surfactant model and to understand the underlying mechanisms of the nanoparticle interaction with the monolayer.

Development and Characterization of Ultrasound Activated Lipopolyplexes for Enhanced Transfection by Low Frequency Ultrasound in In Vitro Tumor Model.

This work focuses on the development of ultrasound contrast vesicles for ultrasound-mediated enhanced transfection of nucleic acids in the cancer cells and projects its application as a tool for diagnostic imaging. The ultrasound contrast vesicles are stable, anionic, nanoscaled vesicles with ultrasound contrast equivalent to the commercially available SonoVue. These anionic lipid vesicles establish electrostatic interaction with cationic polyplexes based on linear polyethylenimine (22kDa) forming lipopolyplexes with ultrasound contrast. The lipopolyplexes are characterized regarding shape, size, and zeta potential. When exposed to low frequency ultrasound, these carriers show elevated transfection efficiency and reduced cytotoxicity. The effect of post-transfection ultrasound on cellular uptake of lipopolyplexes is also evaluated. An analogous transfection is also observed in the tumor mimicking multicellular 3D spheroid culture of ovarian cancer cells. The emergence of tumor imaging and enhanced gene delivery by medical ultrasound, a noninvasive imaging modality, is considered paving the way for efficient theranostic gene therapy.

Targeted ErbB3 cancer therapy: A synergistic approach to effectively combat cancer.

Surface modification of nanoparticles with aptamer is gaining popularity lately due to its selective targeting and low immunogenicity. In this study, sorafenib tosylate (SFB) was loaded in biodegradable PLGA nanoparticles prepared by solvent evaporation method. The surfaces of drug deprived and drug-loaded particles (PN and PNS, respectively) were coupled with aptamer to target ErbB3 using EDC/NHS chemical modification. Nanoparticles were characterized with regard to their size, shape and chemical composition by dynamic light scattering, atomic force microscopy, FTIR and elemental analysis respectively. To evaluate the particles in vitro cell culture studies were performed. Cell viability assay, pathway analysis and apoptosis assay showed cellular toxicity in the presence of aptamer in PNS-Apt (p < 0.001). Metastatic progression assay showed decreased cell migration in the presence of aptamer and SFB. Confocal laser scanning microscopy was used to visualize the receptor-mediated time-dependent intracellular uptake and distribution of the nanoparticles throughout the cytoplasm. The findings of the current study demonstrated the potential efficacy of the surface modified SFB-loaded particles against ErbB3.

Efficient Transfection of Large Plasmids Encoding HIV-1 into Human Cells-A High Potential Transfection System Based on a Peptide Mimicking Cationic Lipid.

One major disadvantage of nucleic acid delivery systems is the low transfection or transduction efficiency of large-sized plasmids into cells. In this communication, we demonstrate the efficient transfection of a 15.5 kb green fluorescent protein (GFP)-fused HIV-1 molecular clone with a nucleic acid delivery system prepared from the highly potent peptide-mimicking cationic lipid OH4 in a mixture with the phospholipid DOPE (co-lipid). For the transfection, liposomes were loaded using a large-sized plasmid (15.5 kb), which encodes a replication-competent HIV type 1 molecular clone that carries a Gag-internal green fluorescent protein (HIV-1 JR-FL Gag-iGFP). The particle size and charge of the generated nanocarriers with 15.5 kb were compared to those of a standardized 4.7 kb plasmid formulation. Stable, small-sized lipoplexes could be generated independently of the length of the used DNA. The transfer of fluorescently labeled pDNA-HIV1-Gag-iGFP in HEK293T cells was monitored using confocal laser scanning microscopy (cLSM). After efficient plasmid delivery, virus particles were detectable as budding structures on the plasma membrane. Moreover, we observed a randomized distribution of fluorescently labeled lipids over the plasma membrane. Obviously, a significant exchange of lipids between the drug delivery system and the cellular membranes occurs, which hints toward a fusion process. The mechanism of membrane fusion for the internalization of lipid-based drug delivery systems into cells is still a frequently discussed topic.

Photodynamic inactivation of circulating tumor cells: An innovative approach against metastatic cancer.

The spread of a primary malignant tumor is the major reason for most of the cancer-associated deaths. To this day, treatment regimen and available drugs are still insufficient to manage these conditions. In this work, a new therapeutic concept based on photodynamic therapy (PDT) of metastasis-initiating cells is introduced. To address this issue, an experimental model was developed to simulate the movement and photodynamic inactivation of circulating tumor cells (CTCs) in vitro. Using curcumin loaded poly(lactic-co-glycolic acid) nanoparticles, a significant reduction in the cell viability of human breast cancer cells (MDA-MB-231) could be achieved after 30 min laser irradiation (λ = 447 nm, P = 100mW) under flow conditions (5 cm s-1). Confocal laser scanning microscopy images confirmed the immediate accumulation of curcumin on the cell membrane and an increased fluorescence signal after irradiation. PDT caused time-dependent morphological cell alterations (i.e. membrane evaginations and disruption) indicating apoptosis and early necrosis. During the photoactivation of curcumin, a blue shift in the absorption spectra and a decrease in the curcumin content could be determined. This study confirms that the presented experimental model is suitable for in vitro investigations of CTCs under in vivo-like conditions, at the same time encouraging the clinical implementation of PDT as an innovative strategy against metastasis.

Lipodendriplexes mediated enhanced gene delivery: a cellular to pre-clinical investigation.

Clinical success of effective gene therapy is mainly hampered by the insufficiency of safe and efficient internalization of a transgene to the targeted cellular site. Therefore, the development of a safe and efficient nanocarrier system is one of the fundamental challenges to transfer the therapeutic genes to the diseased cells. Polyamidoamine (PAMAM) dendrimer has been used as an efficient non-viral gene vector (dendriplexes) but the toxicity and unusual biodistribution induced by the terminal amino groups (-NH2) limit its in vivo applications. Hence, a state of the art lipid modification with PAMAM based gene carrier (lipodendriplexes) was planned to investigate theirs in vitro (2D and 3D cell culture) and in vivo behaviour. In vitro pDNA transfection, lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) generation, cellular protein contents, live/dead staining and apoptosis were studied in 2D cell culture of HEK-293 cells while GFP transfection, 3D cell viability and live/dead staining of spheroids were performed in its 3D cell culture. Acute toxicity studies including organ to body index ratio, hematological parameters, serum biochemistry, histopathological profiles and in vivo transgene expression were assessed in female BALB/c mice. The results suggested that, in comparison to dendriplexes the lipodendriplexes exhibited significant improvement of pDNA transfection (p < 0.001) with lower LDH release (p < 0.01) and ROS generation (p < 0.05). A substantially higher cellular protein content (p < 0.01) and cell viability were also observed in 2D culture. A strong GFP expression with an improved cell viability profile (p < 0.05) was indicated in lipodendriplexes treated 3D spheroids. In vivo archives showed the superiority of lipid-modified nanocarrier system, depicted a significant increase in green fluorescent protein (GFP) expression in the lungs (p < 0.01), heart (p < 0.001), liver (p < 0.001) and kidneys (p < 0.001) with improved serum biochemistry and hematological profile as compared to unmodified dendriplexes. No tissue necrosis was evident in the animal groups treated with lipid-shielded molecules. Therefore, a non-covalent conjugation of lipids with PAMAM based carrier system could be considered as a promising approach for an efficient and biocompatible gene delivery system.

Downregulation of MDR 1 gene contributes to tyrosine kinase inhibitor induce apoptosis and reduction in tumor metastasis: A gravity to space investigation.

P-glycoprotein (P-gp) associated multidrug resistance (MDR) represents a major failure in cancer treatment. The overexpression of P-gp is responsible for ATP-dependent efflux of drugs that decrease their intracellular accumulation. An effective downregulation of MDR1 gene using small interfering RNA (siRNA) is one of the safe and effective tools to overcome the P-gp triggered MDR. Therefore, the development of an efficient and non-toxic carrier system for siRNA delivery is a fundamental challenge for effective cancer treatment. Polyamidoamine (PAMAM) dendrimer has been used for efficient delivery of siRNA (dendriplexes) to the tumor cells but the associated toxicity problems render its use in biological applications. A non-covalent lipid modification (lipodendriplexes) is supposed to offer a promising strategy to overcome the demerits linked to the naked dendriplexes system. In the current study, we deliver siRNA, designed against MDR1 gene (si-MDR1), in colorectal carcinoma cells (Caco-2), having overexpression of P-gp, to check the role of MDR1 gene in tumor progression and multidrug resistance using two dimensional (2D) and three dimensional (3D) environment. Imatinib mesylate (IM), a P-gp substrate, was used as model drug. Our results revealed that the effective knockdown by lipodendriplexes system can significantly reduce the tumor cell migration in 2D (p < 0.001) and 3D (p < 0.001) cell cultures as compared to unmodified dendriplexes and si-Control groups. It was also observed that lipodendriplexes aided downregulation of MDR1 gene effectively, re-sensitized the Caco-2 cells for IM uptake and showed a significantly (p < 0.001) higher apoptosis. Our findings imply that our lipodendriplexes system has a great potential for siRNA delivery, however, further in vivo application using a suitable targeted system can play a major role for better cancer therapeutics.

Magnetic resonance activatable thermosensitive liposomes for controlled doxorubicin delivery.

To limit the massive cytotoxicity of chemotherapeutic agents, it is desirable to establish an appropriate subtle blend of formulation design based on a dual-responsive strategy. In this study, a combined therapeutic platform based on magnetic thermosensitive liposomes (LipTS-GD) was developed. The incorporation of chelated-gadolinium imparted magnetic properties to thermosensitive liposomes (LipTS). The application of an ultra high field magnetic resonance imaging (UHF-MRI) induced hyperthermia, thus provided an improved chemotherapeutic effect of Doxorubicin (DOX). The paramagnetic platform demonstrated thermal sensitivity over a narrow temperature range starting at 37.8 °C, hence the release of DOX from LipTS-GD can be well triggered by inducing hyperthermia using UHF-MRI application. The prepared LipTS-GD were below 200 nm in diameter and an adequate release of DOX reaching 68% was obtained after 1 h UHF-MRI exposure. Profoundly, triple-negative breast cancer (TNBC) cells that were treated with LipTS-GD and subjected thereafter to UHF-MRI exposure for 60 min showed 36% viability. Hemocompatibility studies of LipTS-GD showed a physiological coagulation time and minimal hemolytic potential. Conclusively, LipTS-GD guided local delivery of DOX to solid tumors will potentially raise the therapeutic index, thus reducing the required dose and frequency of DOX administered systemically without influencing the adjacent tissues.

Wavelength dependent photo-cytotoxicity to ovarian carcinoma cells using temoporfin loaded tetraether liposomes as efficient drug delivery system.

5,10,15,20-Tetrakis(3-hydroxyphenyl)chlorin (mTHPC; temoporfin) is one of the most potent second-generation photosensitizers available today for the treatment of a variety of clinical disorders and has a unique capability of being activated at different wavelengths. However, due to its highly lipophilic nature, poor solubility in the aqueous media and poor bioavailability limits its application in anticancer therapies. To overcome these potential issues, we developed three different liposomal formulations with mTHPC encapsulated in hydrophobic milieu thus increasing the bioavailability of the drug. The prepared formulations were characterized in terms of hydrodynamic diameter, surface charge, encapsulation efficiency, and stability studies. The mean size of the liposomes was found to be in the nanoscale range (about 100 nm) with zeta potential ranging from -6.0 to -13.7 mV. mTHPC loaded liposomes were also evaluated for morphology using atomic force microscopy (AFM) and cryo-transmission electron microscopy (cryo-TEM). Data obtained from the hemocompatibility experiments showed that these formulations were compatible with blood showing less than 10% hemolysis and coagulation time lower than 40 s. The results obtained from the single-cell gel electrophoresis assay also demonstrated no incidence of genotoxicity. Photodynamic destruction of SK-OV-3 cells using mTHPC loaded liposomes showed a dose-response relationship upon irradiation with two different wavelength lights (blue λ = 457 nm & red λ = 652 nm). A 10-fold pronounced effect was produced when liposomal formulations were irradiated at 652 nm as compared to 457 nm. This was also evaluated by the quantitative assessment of reactive oxygen production (ROS) using fluorescence microscopy. The qualitative assessment of PDT pre- and post-irradiation was visualized using confocal laser scanning microscopy (CLSM) which demonstrated an intense localization of mTHPC liposomes in the perinuclear region. Chick chorioallantoic membrane assay (CAM) was used as an alternative in-ovo model to demonstrate the localized destruction of tumor microvasculature. Overall, the prepared nanoformulation is a biocompatible, efficient and well characterized delivery system for mTHPC for the safe and effective PDT.

Contact-Triggered Lipofection from Multilayer Films Designed as Surfaces for in Situ Transfection Strategies in Tissue Engineering.

Biomaterials, which release active compounds after implantation, are an essential tool for targeted regenerative medicine. In this study, thin multilayer films loaded with lipid/DNA complexes (lipoplexes) were designed as surface coatings for in situ transfection applicable in tissue engineering and regenerative medicine. The film production and embedding of lipoplexes were based on the layer-by-layer (LbL) deposition technique. Hyaluronic acid (HA) and chitosan (CHI) were used as the polyelectrolyte components. The embedded plasmid DNA was complexed using a new designed cationic lipid formulation, namely, OH4/DOPE 1/1, the advantageous characteristics of which have been proven already. Three different methods were tested regarding its efficiency of lipid and DNA deposition. Therefore, several surface specific analytics were used to characterize the LbL formation, the lipid DNA embedding, and the surface characteristics of the multilayer films, such as fluorescence microscopy, surface plasmon resonance spectroscopy, ellipsometry, zeta potential measurements, atomic force microscopy, and scanning electron microscopy. Interaction studies were conducted for optimized lipoplex-loaded polyelectrolyte multilayers (PEMs) that showed an efficient attachment of C2C12 cells on the surface. Furthermore, no acute toxic effects were found in cell culture studies, demonstrating biocompatibility. Cell culture experiments with C2C12 cells, a cell line which is hard to transfect, demonstrated efficient transfection of the reporter gene encoding for green fluorescent protein. In vivo experiments using the chicken embryo chorion allantois membrane animal replacement model showed efficient gene-transferring rates in living complex tissues, although the DNA-loaded films were stored over 6 days under wet and dried conditions. Based on these findings, it can be concluded that OH4/DOPE 1/1 lipoplex-loaded PEMs composed of HA and CHI can be an efficient tool for in situ transfection in regenerative medicine.

Curcumin loaded nanoparticles as efficient photoactive formulations against gram-positive and gram-negative bacteria.

The constant increase in multi-resistant bacterial strains and the decline in the number of newly approved antibiotics necessitate the development of alternative approaches to antibiotic treatment. In this study, a modern alternative approach to antibiotic therapy using photosensitiser encapsulated polymeric nanoparticles is presented. Cationic nanoparticles were prepared using a biodegradable and biocompatible polymer poly (lactic-co-glycolic acid), a stabiliser poly (vinyl alcohol) and chitosan. Dynamic light scattering and laser Doppler anemometry were used to determine particle size distribution and ζ-potential respectively. To quantify the antibacterial photodynamic effect of the nanoparticles, in vitro studies were performed using Staphylococcus saprophyticus subsp. bovis and Escherichia coli DH5 alpha to represent both a gram-positive as well as a gram-negative strain. It was demonstrated that the particle ζ-potential significantly influenced the antibacterial phototoxicity, gaining up to 3 log10 higher efficacy for chitosan coated nanoparticles. Furthermore, neither irradiation alone nor curcumin in absence of light led to a significant growth reduction, confirming the photodynamic effect of curcumin. Electron microscopy has been used to study the morphological characteristics of the nanoparticles as well as their interaction with bacteria and the changes of bacterial morphology and ultrastructure upon photodynamic treatment. An increased adherence of the chitosan modified nanoparticles to the bacteria and structural damage upon photodynamic treatment was clearly evident and confirmed the results from in vitro studies.