Nanobody-liposomes as novel cancer vaccine platform to efficiently stimulate T cell immunity.

Cancer vaccines can be utilized in combination with checkpoint inhibitors to optimally stimulate the anti-tumor immune response. Uptake of vaccine antigen by antigen presenting cells (APCs) is a prerequisite for T cell priming, but often relies on non-specific mechanisms. Here, we have developed a novel vaccination strategy consisting of cancer antigen-containing liposomes conjugated with CD169- or DC-SIGN-specific nanobodies (single domain antibodies) to achieve specific uptake by APCs. Our studies demonstrate efficient nanobody liposome uptake by human and murine CD169+ and DC-SIGN+ APCs in vitro and in vivo when compared to control liposomes or liposomes with natural ligands for CD169 and DC-SIGN. Uptake of CD169 nanobody liposomes resulted in increased T cell activation by human APCs and stimulated naive T cell priming in mouse models. In conclusion, while nanobody liposomes have previously been utilized to direct drugs to tumors, here we show that nanobody liposomes can be applied as vaccination strategy that can be extended to other receptors on APCs in order to elicit a potent immune response against tumor antigens.

Osteoimmunomodulatory GelMA/liposome coatings to promote bone regeneration of orthopedic implants.

Despite being the most widely used biomaterials in orthopedic surgery, metallic implants do not induce new bone growth because they are bioinert. Surface biofunctionalization of implants with immunomodulatory mediators is a recent approach to promote osteogenic factors that facilitate bone regeneration. Liposomes (Lip) can be used as a low-cost, efficient and simple immunomodulator to stimulate immune cells in favor of bone regeneration. Even though liposomal coating systems have been reported previously, their main disadvantage is their limited ability to preserve liposome integrity after drying. In order to address this issue, we developed a hybrid system in which liposomes could be embedded in a polymeric hydrogel namely gelatin methacryloyl (GelMA). Specifically, we have developed a novel versatile coating strategy using electrospray technology to coat implants with GelMA/Liposome without using adhesive intermediate layer. The two differently charged Lip (i.e., anionic and cationic) were blended with GelMA and coated via electrospray technology on the bone-implant surfaces. The results showed that the developed coating withstood mechanical stress during surgical replacement, and Lip inside GelMA coating stayed intact in different storage conditions for a minimum of 4 weeks. Surprisingly, bare Lip, either cationic or anionic, improved the osteogenesis of human Mesenchymal Stem Cells (MSCs) by inducing pro-inflammatory cytokines, even at a low dosage of Lip released from the GelMA coating. More importantly, we showed that the inflammatory response could be fine-tuned by selecting the Lip concentration, Lip/hydrogel ratio, and coating thickness to determine the timing of the release such that we can accommodate different clinical needs. These promising results pave the way to use these Lip coatings to load different types of therapeutic cargo for bone-implant applications.

Liposome induction of CD8+ T cell responses depends on CD169+ macrophages and Batf3-dependent dendritic cells and is enhanced by GM3 inclusion.

Cancer vaccines aim to efficiently prime cytotoxic CD8+ T cell responses which can be achieved by vaccine targeting to dendritic cells. CD169+ macrophages have been shown to transfer antigen to dendritic cells and could act as an alternative target for cancer vaccines. Here, we evaluated liposomes containing the CD169/Siglec-1 binding ligand, ganglioside GM3, and the non-binding ligand, ganglioside GM1, for their capacity to target antigens to CD169+ macrophages and to induce immune responses. CD169+ macrophages demonstrated specific uptake of GM3 liposomes in vitro and in vivo that was dependent on a functional CD169 receptor. Robust antigen-specific CD8+ and CD4+ T and B cell responses were observed upon intravenous administration of GM3 liposomes containing the model antigen ovalbumin in the presence of adjuvant. Immunization of B16-OVA tumor bearing mice with all liposomes resulted in delayed tumor growth and improved survival. The absence of CD169+ macrophages, functional CD169 molecules, and cross-presenting Batf3-dependent dendritic cells (cDC1s) significantly impaired CD8+ T cell responses, while B cell responses were less affected. In conclusion, we demonstrate that inclusion of GM3 in liposomes enhance immune responses and that splenic CD169+ macrophages and cDC1s are required for induction of CD8+ T cell immunity after liposomal vaccination.

Port wine stain treatment outcomes have not improved over the past three decades.

BACKGROUND: Since the early '80s, the pulsed dye laser has been the standard treatment tool for non-invasive port wine stain (PWS) removal. In the last three decades, a considerable amount of research has been conducted to improve clinical outcomes, given that a fraction of PWS patients proved recalcitrant to laser treatment. Whether this research actually led to increased therapeutic efficacy has not been systematically investigated. OBJECTIVE: To analyse therapeutic efficacy in PWS patients globally from 1986 to date. METHODS: PubMed was searched for all available PWS trials. Studies with a quartile percentage improvement scale were included, analysed and plotted chronologically. Treatment and patient characteristics were extracted. A mean clearance per study was calculated and plotted. A 5-study simple moving average was co-plotted to portray the trend in mean clearance over time. The data were separately analysed for multiple treatment sessions in previously untreated patients. RESULTS: Sixty-five studies were included (24.3% of eligible studies) comprising 6207 PWS patients. Of all patients, 21% achieved 75-100% clearance. Although a few studies reported remarkably good outcomes in a subset of carefully selected patients, there was no upward trend over time in mean clearance. CONCLUSION: The efficacy of PWS therapy has not improved in the past decades, despite numerous technical innovations and pharmacological interventions. With an unwavering patient demand for better outcomes, the need for development and implementation of novel therapeutic strategies to clear all PWS is as valid today as it was 30 years ago.

A technical protocol for an experimental ex vivo model using arterially perfused porcine eyes.

Ex vivo ocular perfused models have been described in the past and were applied in different mammalian species as platforms to test drug delivery systems and surgical techniques. However, reproduction of those methods is challenging because extensive and precise description of the protocols used is lacking. In this technical paper we provide a detailed description of all the steps to be followed from the enucleation of porcine eyes to cannulation of the ophthalmic artery and perfusion. This model can contribute to the reduction of use of living animals in ophthalmology research, whereas as opposed to in vitro models, it preserves tissue complexity and integrity.

Relationship between polarities of antibiotic and polymer matrix on nanoparticle formulations based on aliphatic polyesters.

In the field of nanomedicine, nanoparticles are developed to target antibiotics to sites of bacterial infection thus enabling adequate drug exposure and decrease development of resistant bacteria. In the present study, we investigated the encapsulation of two antibiotics with different polarity into different PEGylated polymeric nanoparticles based on aliphatic polyesters, to obtain a better understanding of critical factors determining encapsulation and release. The nanoparticles were prepared from diblock copolymers comprising of a poly(ethylene glycol) block attached to an aliphatic polyester block of varying polarity: poly(lactic-co-glycolic acid) (mPEG-PLGA), poly(lactic-co-hydroxymethyl glycolic acid) (mPEG-PLHMGA) and poly(lactic-co-benzyloxymethyl glycolic acid) (mPEG-PLBMGA). Hydrophobic bedaquiline and hydrophilic vancomycin were encapsulated via single and double-emulsion solvent evaporation techniques, respectively. Encapsulation, degradation and release studies at physiological simulating conditions were performed. Drug polarity and preparation techniques influenced encapsulation efficiency into polymer nanoparticles, giving almost complete encapsulation of bedaquiline and approx. 30% for vancomycin independent of the polymer type. The nonpolar bedaquiline showed a predominantly diffusion-controlled release independent of polymer composition. However, polar vancomycin was released by a combination of diffusion and polymer degradation, which was significantly affected by polymer composition, the most hydrophilic polymer displaying the fastest release.

Dynamic Fluorescence Microscopy of Cellular Uptake of Intercalating Model Drugs by Ultrasound-Activated Microbubbles.

PURPOSE: The combination of ultrasound and microbubbles can facilitate cellular uptake of (model) drugs via transient permeabilization of the cell membrane. By using fluorescent molecules, this process can be studied conveniently with confocal fluorescence microscopy. This study aimed to investigate the relation between cellular uptake and fluorescence intensity increase of intercalating model drugs. PROCEDURES: SYTOX Green, an intercalating fluorescent dye that displays >500-fold fluorescence enhancement upon binding to nucleic acids, was used as a model drug for ultrasound-induced cellular uptake. SYTOX Green uptake was monitored in high spatiotemporal resolution to qualitatively assess the relation between uptake and fluorescence intensity in individual cells. In addition, the kinetics of fluorescence enhancement were studied as a function of experimental parameters, in particular, laser duty cycle (DC), SYTOX Green concentration and cell line. RESULTS: Ultrasound-induced intracellular SYTOX Green uptake resulted in local fluorescence enhancement, spreading throughout the cell and ultimately accumulating in the nucleus during the 9-min acquisition. The temporal evolution of SYTOX Green fluorescence was substantially influenced by laser duty cycle: continuous laser (100 % DC) induced a 6.4-fold higher photobleaching compared to pulsed laser (3.3 % DC), thus overestimating the fluorescence kinetics. A positive correlation of fluorescence kinetics and SYTOX Green concentration was found, increasing from 0.6 × 10-3 to 2.2 × 10-3 s-1 for 1 and 20 µM, respectively. Finally, C6 cells displayed a 2.4-fold higher fluorescence rate constant than FaDu cells. CONCLUSIONS: These data show that the temporal behavior of intracellular SYTOX Green fluorescence enhancement depends substantially on nuclear accumulation and not just on cellular uptake. In addition, it is strongly influenced by the experimental conditions, such as the laser duty cycle, SYTOX Green concentration, and cell line.

Strategies for the Activation and Release of the Membranolytic Peptide Melittin from Liposomes Using Endosomal pH as a Trigger.

Endosomolytic peptides are often coupled to drug delivery systems to enhance endosomal escape, which is crucial for the delivery of macromolecular drugs that are vulnerable to degradation in the endolysosomal pathway. Melittin is a 26 amino acid peptide derived from bee venom that has a very high membranolytic activity. However, such lytic peptides also impose a significant safety risk when applied in vivo as they often have similar activity against red blood cells and other nontarget cell membranes. Our aim is to control the membrane-disrupting capacity of these peptides in time and space by physically constraining them to a nanocarrier surface in such a way that they only become activated when delivered inside acidic endosomes. To this end, a variety of chemical approaches for the coupling of lytic peptides to liposomes via functionalized PEG-lipids were explored, including maleimide-thiol chemistry, click-chemistry, and aldehyde-hydrazide chemistry. The latter enables reversible conjugation via a hydrazone bond, allowing for release of the peptide under endosomal conditions. By carefully choosing the conjugation site and by using a pH activated analog of the melittin peptide, lytic activity toward a model membrane is completely inhibited at physiological pH. At endosomal pH the activity is restored by hydrolysis of the acid-labile hydrazone bond, releasing the peptide in its most active, free form. Furthermore, using an analogue containing a nonhydrolyzable bond as a control, it was shown that the activity observed can be completely attributed to release of the peptide, validating dynamic covalent conjugation as a suitable strategy to maintain safety during circulation.

Formulation and characterization of microspheres loaded with imatinib for sustained delivery.

The aim of this study was the development of imatinib-loaded poly(d,l-lactide-co-glycolide) (PLGA) microspheres with high loading efficiency which can afford continuous release of imatinib over a prolonged period of time. Imatinib mesylate loaded PLGA microspheres with a size of 6-20 µm were prepared by a double emulsion (W1/O/W2) method using dichloromethane as volatile solvent. It was found that the microspheres were spherical with a non-porous surface; imatinib loading efficiency (LE) was highly dependent on the pH of the external water phase (W2). By increasing the pH of W2 phase above the highest pKa of imatinib (pKa 8.1), at which imatinib is mainly uncharged, the LE increased from 10% to 90% (pH 5.0 versus pH 9.0). Conversely, only 4% of its counter ion, mesylate, was retained in the microspheres at the same condition (pH 9.0). Since mesylate is highly water soluble, it is unlikely that it partitions into the organic phase. We demonstrated, using differential scanning calorimetry (DSC), that imatinib was molecularly dispersed in the polymeric matrix at loadings up to 8.0%. At higher drug loading, imatinib partially crystallized in the matrix. Imatinib microspheres released their cargo during three months by a combination of diffusion through the polymer matrix and polymer erosion. In conclusion, we have formulated imatinib microspheres with high LE and LC. Although we started with a double emulsion of imatinib mesylate, the obtained microspheres contained imatinib base which was mainly molecularly dispersed in the polymer matrix. These microspheres release imatinib over a 3-month period which is of interest for local treatment of cancer.

Sunitinib microspheres based on [PDLLA-PEG-PDLLA]-b-PLLA multi-block copolymers for ocular drug delivery.

Sunitinib is a multi-targeted receptor tyrosine kinase (RTK) inhibitor that blocks several angiogenesis related pathways. The aim of this study was to develop sunitinib-loaded polymeric microspheres that can be used as intravitreal formulation for the treatment of ocular diseases. A series of novel multi-block copolymers composed of amorphous blocks of poly-(D,L-lactide) (PDLLA) and polyethylene glycol (PEG) and of semi-crystalline poly-(L-lactide) (PLLA) blocks were synthesized. Sunitinib-loaded microspheres were prepared by a single emulsion method using dichloromethane as volatile solvent and DMSO as co-solvent. SEM images showed that the prepared microspheres (∼ 30 µm) were spherical with a non-porous surface. Sunitinib-loaded microspheres were studied for their degradation and in-vitro release behavior. It was found that increasing the percentage of amorphous soft blocks from 10% to 30% accelerated the degradation of the multi-block copolymers. Sunitinib microspheres released their cargo for a period of at least 210 days by a combination of diffusion and polymer erosion. The initial burst (release in 24h) and release rate could be tailored by controlling the PEG-content of the multi-block copolymers. Sunitinib-loaded microspheres suppressed angiogenesis in a chicken chorioallantoic membrane (CAM) assay. These microspheres therefore hold promise for long-term suppression of ocular neovascularization.

Intercalating quaternary nicotinamide-based poly(amido amine)s for gene delivery.

In the development of potent polymeric gene carriers for gene therapy, a good interaction between the polymer and the nucleotide is indispensable to form small and stable polyplexes. Polymers with relatively high cationic charge density are frequently used to provide these interactions, but high cationic charge is usually associated with severe cytotoxicity. In this study an alternative, nucleotide specific binding interaction based on intercalation was investigated to improve polymer/pDNA complex formation. For this purpose bioreducible poly(amido amine) copolymers (p(CBA-ABOL/Nic)) were synthesized with different degrees of intercalating quaternary nicotinamide (Nic) groups and amide-substituted derivatives in their side chains. The quaternary nicotinamide group was chosen as intercalating moiety because this group is part of the naturally occurring NAD+ coenzyme and is therefore expected to be non-toxic and non-carcinogenic. The presence of the quaternary nicotinamide moieties in the poly(amido amine) copolymers showed to effectively promote self-assembled polyplex formation already at low polymer/DNA ratios and results in decreased polyplex size and increased stability of the polyplexes. Furthermore, in contrast to the primary amine functionalized analogs the quaternary nicotinamide polymers showed to be non-hemolytic, indicating their compatibility with cell membranes. Polymers with 25% of Nic in the side chains induced GFP expressions of about 4-5 times that of linear PEI, which is comparable with p(CBA-ABOL), the parent PAA without Nic, but at a two- to fourfold lower required polymer dose. N-phenylation of the nicotinamide functionality even further reduces the required polymer dose to form stable polyplexes, which is a major improvement for these kinds of cationic polymers.

Antitumor efficacy of dexamethasone-loaded core-crosslinked polymeric micelles.

In the current study, core-crosslinked polymeric micelles (DEX-PMs) loaded with three different DEX derivatives designed to display different drug release kinetics, were evaluated for cancer therapy and compared to another effective nanomedicine formulation (long-circulating liposomes encapsulating dexamethasone, LCL-DEX). Pharmacokinetic studies with both radiolabeled dexamethasone and polymer showed that these polymeric systems have long circulating half-lives and may accumulate at the tumor site to a higher extent than liposomes. The in vitro drug release profiles and circulating drug levels in the blood stream show that DEX-PMs with dexamethasone covalently entrapped via a sulfone ester-containing linker (DMSL2) have prolonged circulation time and intermediate drug release kinetics compared to the other polymeric DEX-releasing systems. Furthermore, as the free dexamethasone circulating levels were similar when administered as DMSL2-PM or LCL-DEX, these systems were evaluated simultaneously for antitumor efficacy in B16F10 melanoma bearing mice. The corticosteroid-targeted systems inhibited tumor growth to a similar extent and both increased survival compared to free drug. Recently antitumor efficacy of targeted formulations has been correlated with a systemic effect: a decrease of white blood cell count. In this study all three polymeric systems, liposomes as well as free drug had similar effects on the number of circulating white blood cells, although white blood cell counts recovered faster in the group receiving free drug. In conclusion, corticosteroid-targeting with a polymeric system or a liposomal system translates in similar therapeutic effects. The proven high versatility of the PM with possible optimization and adjustment of the drug release to that required by the therapeutic application, clearly demonstrates the potential of these systems for the treatment of chronic inflammatory diseases including cancer.

Intravenous technetium-99m labelled PEG-liposomes in horses: a safety and biodistribution study.

REASONS FOR PERFORMING STUDY: Liposomes are phospholipid nanoparticles that extravasate at sites of increased vascular permeability. They have potential in equine medicine for targeted drug delivery and diagnostic imaging of infectious, inflammatory and neoplastic lesions. OBJECTIVES: This study evaluates the safety and biodistribution of i.v. polyethyleneglycol(PEG) liposomes in normal horses. METHODS: PEG-liposomes were prepared by the film hydration method and labelled using (99m) Tc-hexamethyl-propylene-amine-oxime. A single dose of 0.24 µmol/kg bwt (99m) Tc-PEG-liposomes and 2.4 µmol/kg bwt unlabelled PEG-liposomes was administered to 10 conscious horses via i.v. infusion at a rate of 6 µmol/min for the first 15 min and 60 µmol/min thereafter. Clinical parameters, haematology, plasma biochemistry and serum complement activity were monitored serially. Scintigraphic imaging was performed at 1, 12 and 21 h post infusion (PI). Six horses were subjected to euthanasia at 24 h PI. The percentage injected dose per kilogram of tissue was calculated for multiple organs. Results were analysed using repeated measures ANOVA. RESULTS: Horses did not demonstrate adverse reactions during or after liposome infusion. There was a significant elevation in heart rate and respiratory rate at 20 and 25 min PI. No significant complement consumption was detected, although a trend for decreased total haemolytic complement values at 20 min PI was present. Scintigraphic studies revealed a prolonged vascular phase that lasted to 21 h PI, with a reproducible pattern of organ distribution. Biodistribution studies revealed the highest concentrations of radiopharmaceutical within the lung, kidney, liver and spleen. CONCLUSIONS: Intravenous liposome administration appears to be safe in horses. When administered in combination with PEG-liposomes, (99m) Tc-PEG-liposomes have long circulating characteristics and a reproducible pattern of organ distribution in horses. POTENTIAL RELEVANCE: Radiolabelled liposomes may be useful for detecting infection, inflammation and neoplasia in the horse. Liposomes have significant potential for targeted drug delivery in the horse. This study establishes the scintigraphic findings and tissue distribution of 99mTc-PEG-liposomes after i.v. administration in healthy horses.

Polymeric carrier systems for siRNA delivery.

RNA interference is a technique to induce sequence-specific gene silencing, but is hampered by inefficient delivery of its mediator, short interfering RNA, into target cells. This review describes recent advances in siRNA delivery using polymeric carrier systems. Structural variations that have been applied to these polymers for optimizing their intracellular trafficking are discussed, as well as strategies for stabilization and targeting to diseased tissues in vivo. Recent findings have highlighted safety issues that need to be taken into account in the design of nanoparticles for clinical application.

Targeted delivery of small interfering RNA to angiogenic endothelial cells with liposome-polycation-DNA particles.

Angiogenesis is an attractive target for cancer therapy, due to its central position in tumor growth and development. Vascular Endothelial Growth Factor (VEGF) and its receptors (VEGFRs) play a key role in the angiogenic process. A promising strategy for targeting VEGF-mediated angiogenesis is RNA interference (RNAi) using short interfering RNA (siRNA). However, for efficacious RNAi a well-designed siRNA delivery system is crucial. Liposome-Polycation-DNA (LPD) particles form a promising system for siRNA delivery to tumors. In order to target angiogenic endothelial cells, LPD particles may be modified with a targeting ligand, such as a cyclic Arg-Gly-Asp (RGD) peptide that specifically binds to integrins expressed on tumor-associated endothelial cells. In the current study, RGD-targeted PEGylated LPD particles containing VEGFR-2 siRNA were prepared and optimized with respect to their size and charge by varying protamine content, carrier DNA content for stronger complexation, and PEGylation density. The size of the optimized particles was around 200 nm and the ζ-potential was approximately +20 mV. The uptake and silencing efficacy of the RGD-targeted PEGylated LPD particles were evaluated in H5V cells (murine endothelial cells) and Human Umbilical Vein Endothelial cells (HUVECs). When compared to non-targeted LPD particles, enhanced uptake and silencing of VEGFR-2 expression was observed for RGD-targeted PEGylated LPD particles. In conclusion, the RGD-targeted PEGylated LPD particles containing VEGFR-2 siRNA presented here may be a promising approach for targeting VEGF-mediated angiogenesis in cancer therapy.

Imatinib-ULS-lysozyme: a proximal tubular cell-targeted conjugate of imatinib for the treatment of renal diseases.

The anticancer drug imatinib is an inhibitor of the platelet-derived growth factor receptor (PDGFR) kinases, which are involved in the pathogenesis of fibrotic diseases. In the current study we investigated the delivery of imatinib to the proximal tubular cells of the kidneys and evaluated the potential antifibrotic effects of imatinib in tubulointerstitial fibrosis. Coupling of imatinib to the low molecular weight protein lysozyme via the platinum (II)-based linker ULS yielded a 0.8:1 drug-carrier conjugate that rapidly accumulated in the proximal tubular cells upon intravenous and intraperitoneal administration. The bioavailability of intraperitoneally administered imatinib-ULS-lysozyme was 100%. Renal imatinib levels persisted for up to 3 days after a single injection of imatinib-ULS-lysozyme. Compared with an equal dose imatinib mesylate, imatinib-ULS-lysozyme resulted in a 30- and 15-fold higher renal exposure of imatinib, for intravenous and intraperitoneal administration respectively. Imatinib-ULS-lysozyme could not be detected in the heart, which is the organ at risk for side-effects of prolonged treatment with imatinib. The efficacy of imatinib-ULS-lysozyme in the treatment of tubulointerstitial fibrosis was evaluated in the unilateral ureteral obstruction (UUO) model in mice. Three days UUO resulted in all signs of early fibrosis, i.e. an increased deposition of matrix and production of profibrotic factors. Although a moderately increased activity of PDGFR-ß was observed, the profibrotic phenotype could not be inhibited with imatinib mesylate or with imatinib-ULS-lysozyme. Further evaluation of imatinib mesylate and imatinib-ULS-lysozyme is therefore warranted in an animal model of renal disease in which the activation of PDGFR-ß is more pronounced.

Synthesis and characterization of biodegradable and thermosensitive polymeric micelles with covalently bound doxorubicin-glucuronide prodrug via click chemistry.

Doxorubicin is an anthracycline anticancer agent that is commonly used in the treatment of a variety of cancers, but its application is associated with severe side effects. Biodegradable and thermosensitive polymeric micelles based on poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide-lactate] (mPEG-b-p(HPMAmLac(n))) have been studied as drug delivery systems for therapeutic and imaging agents and have shown promising in vitro and in vivo results. The purpose of this study was to investigate the covalent coupling of a doxorubicin-glucuronide prodrug (DOX-propGA3) to the core of mPEG-b-p(HPMAmLac(2)) micelles. This prodrug is specifically activated by human ß-glucuronidase, an enzyme that is overexpressed in necrotic tumor areas. To this end, an azide modified block copolymer (mPEG(5000)-b-p(HPMAmLac(2)-r-AzEMA)) was synthesized and characterized, and DOX-propGA3 was coupled to the polymer via click chemistry with a high (95%) coupling efficiency. Micelles formed by this DOX containing polymer were small (50 nm) and monodisperse and released 40% of the drug payload after 5 days incubation at 37 °C in the presence of ß-glucuronidase, but less than 5% in the absence of the enzyme. In vitro cytotoxicity experiments demonstrated that DOX micelles incubated with 14C cells showed the same cytotoxicity as free DOX only in the presence of ß-glucuronidase, indicating full conversion of the polymer-bound DOX into the parent drug. Overall, this novel system is very promising for enzymatically responsive anticancer therapy.

An in vitro assay based on surface plasmon resonance to predict the in vivo circulation kinetics of liposomes.

The adsorption of blood proteins onto liposomes and other colloidal particles is an important process influencing the circulation time. Proteins adsorbed to the surface of liposomes can mediate recognition of the liposomes by macrophages of the reticuloendothelial system (RES) facilitating their clearance from the circulation. Coating liposomes with poly(ethylene glycol) (PEG) decreases the blood clearance considerably, most likely due to reduced protein adsorption and/or liposome aggregation. By using the relation between clearance and protein binding, the present study introduces an in vitro assay measuring interactions of liposomes with proteins to predict their blood clearance in vivo. Such assay is valuable since it limits time and costs, and importantly reduces the number of animals required for pharmacokinetic investigations of new formulations. In the current study, Surface Plasmon Resonance (SPR) and fluorescence Single Particle Tracking (fSPT) were used to study liposome-protein interactions and blood induced liposome aggregation in vitro. By means of SPR the interactions between proteins and liposomes coated with PEG of different molecular weights and at different densities (PEG(2000) in 2.5%, 5% and 7%; PEG(5000) in 0.5%, 1.5% and 2.5%), were measured for several plasma proteins: human serum albumin (HSA), apolipoprotein E (ApoE), α2-macroglobulin (α2-M), ß2-glycoprotein (ß2-G) and fibronectin (Fn). Liposomes coated with PEG interacted less with all proteins, an effect which increased with the PEG surface density. In parallel, fSPT analysis showed that the exposure of liposomes to full blood did not change the liposome size, indicating that aggregation is not a strong attributive factor in the clearance of these liposomes. In addition, the SPR measurements of the interactions between liposomes and proteins were correlated with the blood clearance of the liposomes. For each protein, the degree of protein-liposome interaction as determined by SPR showed a moderate to strong positive correlation with the clearance of the liposome type.

Examining the role of Rac1 in tumor angiogenesis and growth: a clinically relevant RNAi-mediated approach.

Angiogenesis, the sprouting of new blood vessels from the pre-existing vasculature, is a well established target in anti-cancer therapy. It is thought that the Rho GTPase Rac1 is required during vascular endothelial growth factor (VEGF)-mediated angiogenesis. In the present study, we have used a clinically relevant RNA interference approach to silence Rac1 expression. Human umbilical vein endothelial cells were transiently transfected with non-specific control siRNA (siNS) or Rac1 siRNA (siRac1) using electroporation or Lipofectamine 2000. Functional assays with transfected endothelial cells were performed to determine the effect of Rac1 knockdown on angiogenesis in vitro. Silencing of Rac1 inhibited VEGF-mediated tube formation, cell migration, invasion and proliferation. In addition, treatment with Rac1 siRNA inhibited angiogenesis in an in vivo Matrigel plug assay. Intratumoral injections of siRac1 almost completely inhibited the growth of grafted Neuro2a tumors and reduced tumor angiogenesis. Together, these data indicate that Rac1 is an important regulator of VEGF-mediated angiogenesis. Knockdown of Rac1 may represent an attractive approach to inhibit tumor angiogenesis and growth.

Liposomal targeting of glucocorticoids to the inflamed synovium inhibits cartilage matrix destruction during murine antigen-induced arthritis.

UNLABELLED: Encapsulation of glucocorticoids into long-circulating liposomes provides targeting of these drugs to the inflamed synovium in experimental arthritis and thereby strongly improves their therapeutic index. The goal of this study was to evaluate the effect and mechanisms of intravenous liposomal delivery of prednisolone phosphate (Lip-PLP) on protease mediated cartilage destruction during murine antigen-induced arthritis (AIA). Mice treated with a single injection of Lip-PLP showed a pronounced suppression of synovial immune cell infiltration compared to control, PBS-treated mice. Liposomal PLP also significantly suppressed interleukin 1ß (3.6 fold) in the synovium, but not in the blood serum. Furthermore, expression of the proteases MMP-3, -9, -13 and -14 and ADAMTS-4 and -5 was suppressed by Lip-PLP in the synovium, but not within the articular cartilage of the femur and tibia, demonstrating the specific action of Lip-PLP on the synovium. Lip-PLP is phagocytosed by macrophages in vitro and suppresses their expression of IL-1ß and MMPs after LPS activation. Moreover, Lip-PLP suppresses destruction of the cartilage matrix during AIA mediated by active MMPs and ADAMTS, as assessed by immunostaining of their respective neoepitopes VDIPEN and NITEGE in various cartilage layers of the knee joint. CONCLUSION: liposomal delivery of glucocorticoids protects against cartilage matrix destruction during experimental arthritis by inhibiting protease expression and activity in the inflamed synovium.