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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

A triple chain polycationic peptide-mimicking amphiphile - efficient DNA-transfer without co-lipids.

Non-viral gene delivery in its current form is largely dependent upon the ability of a delivery vehicle to protect its cargo in the extracellular environment and release it efficiently inside the target cell. Also a simple delivery system is required to simplify a GMP conform production if a marketing authorization is striven for. This work addresses these problems. We have developed a synthetic polycationic peptide-mimicking amphiphile, namely DiTT4, which shows efficient transfection rates and good biocompatibility without the use of a co-lipid in the formulation. The lipid-nucleic acid complex (lipoplex) was characterized at the structural (electron microscopy), physical (laser Doppler velocimetry and atomic force microscopy) and molecular levels (X-ray scattering). Stability studies of the lipoplexes in the presence of serum and heparin indicated a stable formation capable of protecting the cargo against the extracellular milieu. Hemocompatibility studies (hemolysis, complement activation and erythrocyte aggregation) demonstrated the biocompatibility of the formulation for systemic administration. The transfection efficiency was assessed in vitro using the GFP assay and confocal laser scanning microscopy studies. With the chorioallantoic membrane model, an animal replacement model according to the 3R strategy (replacement, refinement, and reduction), initial in vivo experiments were performed which demonstrate fast and efficient transfection in complex tissues and excellent biocompatibility.

Selective anti-ErbB3 aptamer modified sorafenib microparticles: In vitro and in vivo toxicity assessment.

The delivery of aptamer modified therapeutic moieties to specific tissue sites has become one of the major therapeutic choices to reduce the toxicity of inhibitory drugs. Bearing this in mind, the current study was designed using sorafenib (SFB) encapsulated microparticles (MP) prepared with biodegradable poly (D, L-lactic-co-glycolic acid) (PLGA) copolymer. The surfaces of these microparticles were modified with RNA aptamer having a binding affinity towards ErbB3 receptors. SFB-loaded MP (MPS) were prepared by o/w solvent evaporation method and the surface was coupled with the amino group of aptamer by EDC/NHS chemistry. Physiochemical investigations were done by dynamic light scattering, scanning electron microscopy and FTIR. In vitro apoptosis assay, cell viability assay and metastatic progression showed a significant decrease (p < 0.001) in vitro cell viability for MPS and MPS-Apt as compared to MP. The synergistic combination of SFB and aptamer also decreased the metastatic progression of cells for an extended period. Microparticles were also evaluated for in vivo toxicity in female BALB/c mice. It was evident that the presence of aptamer decreased the generalized toxicity of MPS-Apt, as measured by mean body weight loss and blood profiles, keeping all the blood formed elements level within acceptable limits. The histopathological investigations showed some necrotic and pyknotic bodies. In a similar fashion, liver function test and renal function tests showed pronounced effects of formulations on vital organs.

Hypericin inclusion complexes encapsulated in liposomes for antimicrobial photodynamic therapy.

The naturally occurring anthraquinone derivative hypericin is a highly potent photosensitiser. Several in vitro studies show high phototoxicity of the pigment towards gram-positive bacteria. Nevertheless, the highly lipophilic nature and poor bioavailability prevent its application in daily clinical practice thus leading to a limited therapeutic value of hypericin. Liposomal encapsulation could help overcome these limitations and would make hypericin available for daily clinical practice. The use of liposomes as carriers for hypericin in antimicrobial photodynamic therapy (aPDT) is quite new. The aim of this work was to improve the photodynamic efficiency of the previously mentioned carriers by entrapping hypericin in the aqueous compartment of the liposomes. Therefore, a water-soluble inclusion complex of hypericin and (2-hydroxypropyl)-beta-cyclodextrin (Hyp-HPßCD) was prepared. After encapsulation of the inclusion complex into DSPC and DSPC/DPPC/DSPE-PEG liposomes with the dehydration-rehydration vesicle (DRV) method, the formulations were physicochemical characterised. The photodynamic efficiency towards the gram-positive model strain Staphylococcus saprophyticus subsp. bovis. was tested on planktonic cells as well as on biofilms. DSPC liposomes achieved a 4.1log reduction and the DSPC/DPPC/DSPE-PEG liposomes a 2.6log reduction in growth of planktonic bacteria, while Hyp-HPßCD showed total eradication. Even bacterial cells growing in a biofilm could be treated effectively in vitro.

Synergistic effects of ultrasound and photodynamic therapy leading to biofilm eradication on polyurethane catheter surfaces modified with hypericin nanoformulations.

Catheter related infections are causing one third of all blood stream infections. The mortality of those infections is very high and the gold standard for catheter related blood stream infections (CR-BSI) is still the removal of the catheter and systemic antibiotic therapy. There already exist some approaches to prevent the biofilm formation on catheter material, which are far from ideal. A new strategy to prevent bacterial colonization on catheter surfaces is the application of photodynamic therapy (PDT). Therefor the surface has to be modified with substances that can be activated by light, leading to the production of cell toxic reactive oxygen species (ROS). Only small concentrations of the so called photosensitizer (PS) are necessary, avoiding side effects in human therapy. Furthermore, there is no resistance development in PDT. In this study polyurethane (PUR) surfaces were coated with hypericin nanoformulations, leading to 4.3 log10 reduction in bacterial growth in vitro. The effect could be enhanced by the application of ultrasound. The combination of PDT with ultrasound therapy led to a synergistic effect resulting in a 6.8 log10 reduction of viable counts. This minimal invasive method requires only an optical fibre inserted in the catheter lumen and an ultrasound device. Thus the implementation in daily clinical practice is very simple.

The Use of Artificial Gel Forming Bolalipids as Novel Formulations in Antimicrobial and Antifungal Therapy.

The alarming growth of multi-drug resistant bacteria has led to a quest for alternative antibacterial therapeutics. One strategy to circumvent the already existing resistance is the use of photodynamic therapy. Antimicrobial photodynamic therapy (aPDT) involves the use of non-toxic photosensitizers in combination with light and in situ oxygen to generate toxic radical species within the microbial environment which circumvents the resistance building mechanism of the bacteria. Hydrogels are used ubiquitously in the biological and pharmaceutical fields, e.g., for wound dressing material or as drug delivery systems. Hydrogels formed by water-insoluble low-molecular weight gelators may potentially provide the much-needed benefits for these applications. Bolalipids are a superior example of such gelators. In the present work, two artificial bolalipids were used, namely PC-C32-PC and Me2PE-C32-Me2PE, which self-assemble in water into long and flexible nanofibers leading to a gelation of the surrounding solvent. The aim of the study was to create stable hydrogel formulations of both bolalipids and to investigate their applicability as a novel material for drug delivery systems. Furthermore, methylene blue-a well-known photosensitizer-was incorporated into the hydrogels in order to investigate the aPDT for the treatment of skin and mucosal infections using a custom designed LED device.