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.

Co-delivery of carbonic anhydrase IX inhibitor and doxorubicin as a promising approach to address hypoxia-induced chemoresistance.

Hypoxia, an oxygen-deprived condition of the tumor, is one of the major reasons for resistance to chemotherapy. Carbonic anhydrases are generally involved in pH homeostasis in normal conditions, but in solid tumors having a strong relation with hypoxia, the carbonic anhydrase IX (CA-IX) enzyme is overexpressed and results in an extracellular acidic environment. For most weakly basic anticancer drugs, including doxorubicin (Dox), the ionization in an acidic environment limits their cellular uptake, and consequently, the tumor exposure to the drug at sub-therapeutic concentration comes out as chemoresistance. Herein, a combined drug delivery system of liposomes and mesoporous silica nanoparticles (MSNPs) was developed for the co-delivery of the CA-IX enzyme inhibitor and Dox in hypoxic condition. The unique structure of MSNPs with higher surface area was utilized for higher drug loading and sustained release of Dox. Additionally, the biocompatible nature of liposomal coating as a second loading site for the CA-IX enzyme inhibitor has provided gatekeeping effects at pore opening to avoid premature drug release. Lipid coated MSNPs as a co-delivery system for Dox and the CA-IX inhibitor have synergistic cytotoxic effects against MDA-MB 231 breast cancer cells in hypoxic conditions. These findings assure the potential of this drug delivery system to overcome hypoxia-related chemoresistance.

Assessing the Oxidative State of the Skin by Combining Classical Tape Stripping with ORAC Assay.

The antioxidant barrier system of the skin acts as the main defence against environmental pro-oxidants. Impaired skin oxidative state is linked to unhealthy conditions such as skin autoimmune diseases and cancer. Thus, the evaluation of the overall oxidative state of the skin plays a key role in further understanding and prevention of these disorders. This study aims to present a novel ex vivo model to evaluate the skin oxidative state by the measurement of its antioxidant capacity (AOC). For this the ORAC assay was combined with classical tape stripping and infrared densitometry to evaluate the oxidative state of the stratum corneum (SC). Outcomes implied the suitability of the used model to determine the intrinsic antioxidant capacity (iAOC) of the skin. The average iAOC of untreated skin was determined as 140 ± 7.4 µM TE. Skin exposure to UV light for 1 h reduced the iAOC by about 17%, and exposure for 2 h decreased the iAOC by about 30%. Treatment with ascorbic acid (AA) increased the iAOC in a dose-dependent manner and reached an almost two-fold iAOC when 20% AA solution was applied on the skin. The application of coenzyme Q10 resulted in an increase in the iAOC at low doses but decreased the iAOC when doses > 1% were applied on the skin. The results show that the combination of classical tape stripping and ORAC assay is a cost-effective and versatile method to evaluate the skin oxidative state and the pro-oxidate and antioxidative effects of topical skin treatments on the iAOC of the skin. Therefore, the model can be considered to be a valuable tool in skin research.

Cucumber-Derived Exosome-like Vesicles and PlantCrystals for Improved Dermal Drug Delivery.

(1) Background: Extracellular vesicles (EVs) are considered to be efficient nanocarriers for improved drug delivery and can be derived from mammalian or plant cells. Cucumber-derived EVs are not yet described in the literature. Therefore, the aim of this study was to produce and characterize cucumber-derived EVs and to investigate their suitability to improve the dermal penetration efficacy of a lipophilic active ingredient (AI) surrogate. (2) Methods: The EVs were obtained by classical EVs isolation methods and by high pressure homogenization (HPH). They were characterized regarding their physico-chemical and biopharmaceutical properties. (3) Results: Utilization of classical isolation and purification methods for EVs resulted in cucumber-derived EVs. Their dermal penetration efficacy for the AI surrogate was 2-fold higher when compared to a classical formulation and enabled a pronounced transdermal penetration into the viable dermis. HPH resulted in submicron sized particles composed of a mixture of disrupted plant cells. A successful isolation of pure EVs from this mixture was not possible with classical EVs isolation methods. The presence of EVs was, therefore, proven indirectly. For this, the lipophilic drug surrogate was admixed to the cucumber juice either prior to or after HPH. Admixing of the drug surrogate to the cucumber prior to the HPH resulted in a 1.5-fold increase in the dermal penetration efficacy, whereas the addition of the AI surrogate to the cucumber after HPH was not able to improve the penetration efficacy. (4) Conclusions: Results, therefore, indicate that HPH causes the formation of EVs in which AI can be incorporated. The formation of plant EVs by HPH was also indicated by zeta potential analysis.

Assessing the Dermal Penetration Efficacy of Chemical Compounds with the Ex-Vivo Porcine Ear Model.

(1) Background: The ex vivo porcine ear model is often used for the determination of the dermal penetration efficacy of chemical compounds. This study investigated the influence of the post-slaughter storage time of porcine ears on the dermal penetration efficacy of chemical compounds. (2) Methods: Six different formulations (curcumin and different fluorescent dyes in different vehicles and/or nanocarriers) were tested on ears that were (i) freshly obtained, (ii) stored for 24 or 48 h at 4 °C after slaughter before use and (iii) freshly frozen and defrosted 12 h before use. (3) Results: Results showed that porcine ears undergo post-mortem changes. The changes can be linked to rigor mortis and all other well-described phenomena that occur with carcasses after slaughter. The post-mortem changes modify the skin properties of the ears and affect the penetration efficacy. The onset of rigor mortis causes a decrease in the water-holding capacity of the ears, which leads to reduced penetration of chemical compounds. The water-holding capacity increases once the rigor is released and results in an increased penetration efficacy for chemical compounds. Despite different absolute penetration values, no differences in the ranking of penetration efficacies between the different formulations were observed between the differently aged ears. (4) Conclusions: All different types of ears can be regarded to be suitable for dermal penetration testing of chemical compounds. The transepidermal water loss (TEWL) and/or skin hydration of the ears were not correlated with the ex vivo penetration efficacy because both an impaired skin barrier and rigor mortis cause elevated skin hydration and TEWL values but an opposite penetration efficacy. Other additional values (for example, pH and/or autofluorescence of the skin) should, therefore, be used to select suitable and non-suitable skin areas for ex vivo penetration testing. Finally, data from this study confirmed that smartFilms and nanostructured lipid carriers (NLC) represent superior formulation strategies for efficient dermal and transdermal delivery of curcumin.

Thermosensitive liposomes encapsulating hypericin: Characterization and photodynamic efficiency.

The potent photodynamic properties of Hypericin (Hyp) elicit a range of light-dose-dependent anti-tumor activities. However, its low water solubility hampers its broad application. Therefore, the administration of Hyp into biological systems requires drug carriers that would enable sufficient bioavailability. Stimuli-triggered nanocarriers, which are sensitive to endogenous or exogenous stimuli, have become an attractive replacement for conventional therapeutic regimens. Herein, we produced optimized Hyp thermosensitive liposomes (Hyp-TSL), self-assembled from DPPC, DSPC, DSPE-PEG2000. Hyp-TSL displayed a hydrodynamic diameter below 100 nm with an adequate encapsulation efficiency of 94.5 % and good colloidal stability. Hyp-TSL exhibited thermal sensitivity over a narrow range with a phase transition temperature of 41.1 °C, in which liposomal destruction was evident in AFM images after elevated temperature above the phase transition temperature. The uptake of TSL-Hyp into MDA-MB-231 cells was significantly increased with hyperthermic treatment of 42 °C when compared to the uptake at a average physiological temperature of 37 °C. Consequent enhancement of cellular reactive oxygen species was observed after hyperthermic treatment at 42 °C. The half-maximal inhibitory concentration of Hyp TSL was reduced by 3.8 fold after hyperthermic treatment at 42 °C in comparison to treatment at 37 °C. Hyp-TSL were considered safe for intravenous applications as compared by hemocompatibility studies, where coagulation time was <50 s and hemolytic potential was <10%. Conclusively, the enhancement in tumor drug availability correlated with improved therapeutic outcomes.

Ultrasound-Responsive Smart Drug Delivery System of Lipid Coated Mesoporous Silica Nanoparticles.

The immediate release of chemotherapeutics at the target site, along with no premature release in circulation is always challenging. The purpose of this study was to develop a stimuli responsive drug delivery system, composed of lipid supported mesoporous silica nanoparticles (MSNPs) for triggered drug release at the target site and simultaneously avoiding the premature release. MSNPs with a higher drug loading capacity and very slow release were designed so as to enhance release by FDA approved US-irradiation. Doxorubicin, as a model drug, and perfluoropentane (PFP) as a US responsive material, were entrapped in the porous structure of MSNPs. Lipid coating enhanced the cellular uptake and in addition provided a gatekeeping effect at the pore opening to reduce premature release. The mechanical and thermal effects of US induced the conversion of liquid PFP to a gaseous form that was able to rupture the lipid layer, resulting in triggered drug release. The prolonged stability profile and non-toxic behavior made them suitable candidate for the delivery of anticancer drugs. This smart system, with the abilities of better cellular uptake and higher cytotoxic effects on US-irradiation, would be a good addition to the applied side of chemotherapeutic advanced drug delivery systems.

Influence of lipid matrix composition on biopharmaceutical properties of lipid nanoparticles.

Lipid nanoparticles (LN) were invented in the early 1990ties and can be exploited for oral and topical drug delivery to increase the bioavailability of lipophilic active compounds. The lipid matrix of the LN can be composed of solid lipids or of a mixture of liquid and solid lipids. The influence of the lipid matrix composition of LN on the dermal penetration efficacy is not known and was therefore investigated in this study. For this the whole spectrum of LN, that means NE (100% liquid lipid), SLN (100% solid lipid) and NLC that contained low, medium and high amounts of oil were produced and characterized in regard to size, zeta potential, crystallinity and in-vitro release. In addition, the dermal penetration efficacy was determined ex-vivo and the bio-physical skin parameters, i.e., spreadability on skin, skin hydration, skin friction and transepidermal water loss were also assessed. Results demonstrate the tremendous influence of the lipid matrix composition on the biopharmaceutical properties of the LN but showed only minor differences in the physico-chemical properties of the particles. The physico-chemical properties of the LN and the in-vitro release data were not clearly linked to the dermal penetration efficacy, because also other parameters, e.g., skin hydration, spreadability of the formulation on skin and/or film formation of the LN on skin were found to be important parameters that influence the dermal penetration efficacy. Therefore, to allow for the development of effective LN formulations with tailor-made biopharmaceutical properties, not only the physico-chemical properties and in-vitro drug release profiles but also the most relevant biopharmaceutical properties of the LN should be assessed during the formulation development of LN.

Enhanced efficacy and drug delivery with lipid coated mesoporous silica nanoparticles in cancer therapy.

The exposure of cancer cells to subtherapeutic drug concentrations results in multidrug resistance (MDR). The uniqueness of mesoporous silica nanoparticles (MSNPs) with larger surface area for higher drug loading can solve the issue by delivering higher amounts of chemotherapeutics to the cancer cells. However, premature drug release and lower biocompatibility remain challenging. Lipid coating of MSNPs at the same time, can enhance the stability and biocompatibility of nanocarriers. Furthermore, the lipid coating can reduce the systemic drug release and deliver higher amounts to the tumor site. Herein, lipid coated MSNPs were prepared by utilizing cationic liposomes and further investigations were made. Our studies have shown the higher entrapment of doxorubicin (Dox) to MSNPs due to availability of porous structure. Lipid coating could provide a barrier to sustain the release of drug along with reduced premature leakage. In addition, the biocompatibility and enhanced interaction of cationic liposomes to cell membranes resulted in better cellular uptake. Lipid coated silica nanoparticles have shown higher cellular toxicity as compared to non-lipid coated particles. The increase in cytotoxicity with time supports the hypothesis of sustained release of drug from lipid coated MSNPs. We propose the Lip-Dox-MSNPs as an effective approach to treat cancer by delivering and maintaining effective concentration of drugs to the tumor site without systemic side effects.

Improved Antioxidant Capacity of Black Tea Waste Utilizing PlantCrystals.

Antioxidants are recommended to prevent and treat oxidative stress diseases. Plants are a balanced source of natural antioxidants, but the poor solubility of plant active molecules in aqueous media can be a problem for the formulation of pharmaceutical products. The potential of PlantCrystal technology is known to improve the extraction efficacy and antioxidant capacity (AOC) of different plants. However, it is not yet proved for plant waste. Black tea (BT) infusion is consumed worldwide and thus a huge amount of waste occurs as a result. Therefore, BT waste was recycled into PlantCrystals using small-scale bead milling. Their characteristics were compared with the bulk-materials and tea infusion, including particle size and antioxidant capacity (AOC) in-vitro. Waste PlantCrystals possessed a size of about 280 nm. Their AOC increased with decreasing size according to the DPPH (1,1-diphenyl-2-picrylhydrazyl) and ORAC (oxygen radical absorbance capacity) assays. The AOC of the waste increased about nine-fold upon nanonization, leading to a significantly higher AOC than the bulk-waste and showed no significant difference to the infusion and the used standard according to DPPH assay. Based on the results, it is confirmed that the PlantCrystal technology represents a natural, cost-effective plant-waste recycling method and presents an alternative source of antioxidant phenolic compounds.

ADAM 8 as a novel target for doxorubicin delivery to TNBC cells using magnetic thermosensitive liposomes.

Metastatic breast cancer is one of the most common causes of cancer-related death in women worldwide. The transmembrane metalloprotease-disintegrin (ADAM8) protein is highly overexpressed in triple-negative breast cancer (TNBC) cells and potentiates tumor cell invasion and extracellular matrix remodeling. Exploiting the high expression levels of ADAM8 in TNBC cells by delivering anti-ADAM8 antibodies efficiently to the targeted site can be a promising strategy for therapy of TNBC. For instance, a targeted approach with the aid of ultra-high field magnetic resonance imaging (UHF-MRI) activatable thermosensitive liposomes (LipTS-GD) could specifically increase the intracellular accumulation of cytotoxic drugs. The surface of doxorubicin-loaded LipTS-GD was modified by covalent coupling of MAB1031 antibody (LipTS-GD-MAB) in order to target the overexpressed ADAM8 in ADAM8 positive MDA-MB-231 cells. Physicochemical characterization of these liposomes was performed using size, surface morphology and UHF-MRI imaging analysis. In vitro cell targeting was investigated by the washing and circulation method. Intracellular trafficking and lysosomal colocalization were assessed by fluorescence microscopy. Cell viability, biocompatibility and in-ovo CAM assays were performed to determine the effectiveness and safety profiles of liposome formulations. Our results show specific binding and induction of doxorubicin release after LipTS-GD-MAB treatment caused a higher cytotoxic effect at the cellular target site.

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.

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.

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.

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.

Chemotherapy Based on Supramolecular Chemistry: A Promising Strategy in Cancer Therapy.

Chemotherapeutic agents are considered one of the strategies in treating cancer. However, their use is faced by many challenges, such as poor water solubility leading to poor bioavailability and non-selective targeting of cancerous cells leading to diminished therapeutic actions and systemic adverse effects. Many approaches were adopted to overcome these drawbacks and to achieve the targeted delivery of the chemotherapeutic agents to the cancerous cells while minimizing adverse effects. Recently, supramolecular systems such as macrocycles have gained attention in the field of cancer therapy for being able to encapsulate different anticancer drugs via either host-guest complexation or self-assembly leading to a myriad of advantages. This review highlights the most recent studies concerned with the design of such novel systems for cancer therapy.

Nanoenabled Bioseparations: Current Developments and Future Prospects.

The use of nanomaterials in bioseparations has been recently introduced to overcome the drawbacks of the conventional methods. Different forms of nanomaterials, particularly magnetic nanoparticles (MNPs), carbon nanotubes (CNTs), casted nanoporous membranes, and electrospun nanofiber membranes were utilized in biological separation for the aim of production of different biomolecules such as proteins, amino acids, nucleic acids, and enzymes. This paper critically reviews the state-of-the-art efforts undertaken in this regard, with emphasis on the synthesis and performance evaluation of each nanoform. Challenges and future prospects in developing nanoenabled bioseparations are also discussed, for the purpose of highlighting potential advances in the synthesis and fabrication of novel nanomaterials as well as in the design of efficient nanoenabled processes for separating a wide spectrum of biomolecules.

Photodynamic therapy - hypericin tetraether liposome conjugates and their antitumor and antiangiogenic activity.

Photodynamic therapy (PDT) is an established noninvasive tumor treatment. The hydrophobic natural occurring pigment hypericin shows a lot of attractive properties for the application in PDT. Hence, the administration to biological systems or patients requires the formulation in drug carriers enabling sufficient bioavailability. Therefore, free hypericin was encapsulated by the thin film hydration method or a hypericin-hydroxypropyl-ß-cyclodextrin inclusion complex (Hyp-HPßCD) was incorporated by dehydration-rehydration vesicle method in either conventional or ultra-stable tetraether lipid (TEL) liposomes. The hydrodynamic diameter of the prepared nanoformulations ranged between 127 and 212 nm. These results were confirmed by atomic force microscopy. All liposomes showed a good stability under physiological conditions. TEL liposomes which tend to build more rigid bilayers, generate higher encapsulation efficiencies than their conventional counterparts. Furthermore, the suitability for intravenous application was confirmed by hemocompatibility studies resulting in a hemolytic potential less than 20% and a coagulation time less than 50 sec. The uptake of liposomal hypericin into human ovarian carcinoma cells (SK-OV-3) was confirmed using confocal microscopy and further characterized by pathway studies. It was demonstrated that the lipid composition and intraliposomal hypericin localization influenced the anti-vascular effect in the chorioallantoic membrane (CAM). While hypericin TEL liposomes exhibit substantial destruction of the microvasculature drug-in-cyclodextrin TEL liposomes showed no effect. Nevertheless, both formulations yielded severe photocytotoxicity in SK-OV-3 cells in a therapeutic dosage range. Conclusively, hypericin TEL liposomes would be perfectly suited for anti-vascular targeting while Hyp-HPßCD TEL liposomes could deliver the photosensitizer to the tumor site in a more protected manner.

Thrombin-Inhibiting Anticoagulant Liposomes: Development and Characterization.

Many peptides and peptidomimetic drugs suffer from rapid clearance in vivo; this can be reduced by increasing their size through oligomerization or covalent conjugation with polymers. As proof of principle, an alternative strategy for drug oligomerization is described, in which peptidomimetic thrombin inhibitors are incorporated into the liposome surface. For this purpose, the inhibitor moieties were covalently coupled to a palmitic acid residue through a short bifunctionalized ethylene glycol spacer. These molecules were directly added to the lipid mixture used for liposome preparation. The obtained liposomes possess strong thrombin inhibitory potency in enzyme kinetic measurements and anticoagulant activity in plasma. Their strong potency and positive ζ potential indicate that large amounts of the benzamidine-derived inhibitors are located on the surface of the liposomes. This concept should be applicable to other drug molecules that suffer from rapid elimination and allow covalent modification with a suitable fatty acid residue.

Correlation of structure and echogenicity of nanoscaled ultrasound contrast agents in vitro.

Ultrasound is a common tool for clinical diagnosis due to its safety and economic. Especially the addition of ultrasound contrast agents leads to a high diagnostic reliability. In recent years ultrasound has been used as a trigger for directed drug delivery or to enhance thrombolysis. We developed a nanoscaled ultrasound contrast agent (NUSCA) to improve these applications. In the future drugs can be incorporated into this contrast agent to achieve a combination of ultrasound diagnosis and therapy. The aim of the present study is to elucidate the structure of the nanoscaled lipid formulations and a potential dependence of the ultrasound contrast enhancement on this structure. Our NUSCA is based on the phospholipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and the single-chained polyethylene glycol (40) stearate (PEG40S). In this study the effect of increasing concentrations of the single chained PEG40S on the structure of the lipid formulations was characterised using Dynamic Light Scattering, cryo-Transmission Electron Microscopy, Nuclear Magnetic Resonance spectroscopy, lipid monolayer studies and epifluorescence measurements. In addition, the ultrasound contrast enhancement for the formulations was determined in vitro. Dependence between structure and ultrasound contrast was found. All PEG40S concentrations lead to a mixture of liposomes and discoid micelles. With increasing PEG40S content the amount of micelles increased. Certain PEG40S concentrations lead to an ultrasound contrast superior to the contrast of the commercially available ultrasound contrast agent SonoVue(®).