Fast, Efficient, and Targeted Liposome Delivery Mediated by DNA Hybridization.

Safety and efficacy, two significant parameters in drug administration, can be improved by site-specific delivery approaches. Here a fast, efficient, and targeted liposome delivery system steered by a DNA hybridization recognition mechanism is presented. For this purpose, lipid-terminated DNA is inserted in both liposome and cell membranes by simple mixing of the components. Cellular accumulation of cargo encapsulated in the liposomal core is substantially enhanced when the DNA sequence on the cell is complementary to that on the liposome. Additionally, in mixed cell populations, liposomes discriminate targets by their complementary DNA sequences. Exposure of cells to low temperature and endocytosis inhibitors suggests a caveolae-dependent endocytosis uptake pathway. Mechanistically, hybridization between DNA strands spatially traps liposomes and cell membranes in close proximity, consequently increases the local liposome concentration, and thereby enhances cellular uptake of liposomes and their payload. This programmable delivery system might contribute to new applications in molecular biology and drug delivery.

Efficient Fusion of Liposomes by Nucleobase Quadruple-Anchored DNA.

Anchoring DNA via hydrophobic units into the membrane of vesicles allows tagging of these nanocontainers with sequence information. Moreover, the hybridization of DNA on the surface of liposomes enables sequence specific functionalization, vesicle aggregation, and vesicle fusion. Specifically, DNA-hybridization-based approaches for fusion employing oligonucleotides terminally modified with one or two anchoring units were hindered by a limited degree of full fusion or by significant leakage during fusion. The current work deals with a new strategy for anchoring oligonucleotides on a membrane by lipid-modified nucleobases rather than by attaching hydrophobic units to the 3'- or 5'-termini. The lipid anchors were incorporated into the DNA sequence via phosphoramidite nucleotide building blocks during automated solid-phase synthesis allowing variation of the number and position of hydrophobic units along the DNA backbone. Single-stranded DNA functionalized with four lipid-modified nucleobases was stably grafted onto the membrane of lipid vesicles. It was found that the orientation of DNA hybridization and the number of anchoring units play a crucial role in liposomal fusion, which in the most efficient system reached remarkable 29 % content mixing without notable leakage.

Targeting iron metabolism in drug discovery and delivery.

Iron fulfils a central role in many essential biochemical processes in human physiology; thus, proper processing of iron is crucial. Although iron metabolism is subject to relatively strict physiological control, numerous disorders, such as cancer and neurodegenerative diseases, have recently been linked to deregulated iron homeostasis. Consequently, iron metabolism constitutes a promising and largely unexploited therapeutic target for the development of new pharmacological treatments for these diseases. Several iron metabolism-targeted therapies are already under clinical evaluation for haematological disorders, and these and newly developed therapeutic agents are likely to have substantial benefit in the clinical management of iron metabolism-associated diseases, for which few efficacious treatments are currently available.

Liposomal corticosteroids for the treatment of inflammatory disorders and cancer.

Glucocorticoids (GC) are known for their potent immunosuppressive and anti-inflammatory properties. As a consequence, they have been extensively used for the treatment of many different diseases. Prolonged and/or high-dose GC therapy, however, generally comes with severe side effects, resulting not only from their very diverse mechanism(s) of action, but also from their relatively poor biodistribution. Drug delivery systems, and in particular liposomes, have been extensively used to enhance the biodistribution and the target site accumulation of GC, and to thereby improve the balance between their efficacy and their toxicity. Many different types of liposomes have been employed, and both local and systemic treatments have been evaluated. We here summarize the progress made in the use of liposomal GC formulations for the treatment of asthma, rheumatoid arthritis, multiple sclerosis and cancer, and we show that the targeted delivery of GC to pathological sites holds significant clinical potential.

Absolute MR thermometry using nanocarriers.

Accurate time-resolved temperature mapping is crucial for the safe use of hyperthermia-mediated drug delivery. We here propose a magnetic resonance imaging temperature mapping method in which drug delivery systems serve not only to improve tumor targeting, but also as an accurate and absolute nano-thermometer. This method is based on the temperature-dependent chemical shift difference between water protons and the protons in different groups of drug delivery systems. We show that the chemical shift of the protons in the ethylene oxide group in polyethylene glycol (PEG) is temperature-independent, whereas the proton resonance of water decreases with increasing temperature. The frequency difference between both resonances is linear and does not depend on pH and physiological salt conditions. In addition, we show that the proton resonance of the methyl group in N-(2-hydroxypropyl)-methacrylamide (HPMA) is temperature-independent. Therefore, PEGylated liposomes, polymeric mPEG-b-pHPMAm-Lac2 micelles and HPMA copolymers can provide a temperature-independent reference frequency for absolute magnetic resonance (MR) thermometry. Subsequently, we show that multigradient echo MR imaging with PEGylated liposomes in situ allows accurate, time-resolved temperature mapping. In conclusion, nanocarrier materials may serve as highly versatile tools for tumor-targeted drug delivery, acting not only as hyperthermia-responsive drug delivery systems, but also as accurate and precise nano-thermometers.

ß-Thalassemia and Polycythemia vera: targeting chronic stress erythropoiesis.

ß-Thalassemia and Polycythemia vera are genetic disorders which affect the synthesis of red blood cells, also referred to as erythropoiesis. Although essentially different in clinical presentation - patients with ß-thalassemia have an impairment in ß-globin synthesis leading to defective erythrocytes and anemia, while patients with Polycythemia vera present with high hemoglobin levels because of excessive red blood cell synthesis - both pathologies may characterized by lasting high erythropoietic activity, i.e. chronic stress erythropoiesis. In both diseases, therapeutic strategies targeting chronic stress erythropoiesis may improve the address phenotype and prevent secondary pathology, such as iron overload. The current review will address the basic concepts of these strategies to reduce chronic stress erythropoiesis, which may have significant clinical implications in the near future.

Nanomedicines for inflammatory arthritis: head-to-head comparison of glucocorticoid-containing polymers, micelles, and liposomes.

As an emerging research direction, nanomedicine has been increasingly utilized to treat inflammatory diseases. In this head-to-head comparison study, four established nanomedicine formulations of dexamethasone, including liposomes (L-Dex), core-cross-linked micelles (M-Dex), slow releasing polymeric prodrugs (P-Dex-slow), and fast releasing polymeric prodrugs (P-Dex-fast), were evaluated in an adjuvant-induced arthritis rat model with an equivalent dose treatment design. It was found that after a single i.v. injection, the formulations with the slower drug release kinetics (i.e., M-Dex and P-Dex-slow) maintained longer duration of therapeutic activity than those with relatively faster drug release kinetics, resulting in better joint protection. This finding will be instructional in the future development and optimization of nanomedicines for the clinical management of rheumatoid arthritis. The outcome of this study also illustrates the value of such head-to-head comparison studies in translational nanomedicine research.

Distinct roles for hepcidin and interleukin-6 in the recovery from anemia in mice injected with heat-killed Brucella abortus.

Anemia of inflammation (AI) is commonly observed in chronic inflammatory states and may hinder patient recovery and survival. Induction of hepcidin, mediated by interleukin 6, leads to iron-restricted erythropoiesis and anemia. Several translational studies have been directed at neutralizing hepcidin overexpression as a therapeutic strategy against AI. However, additional hepcidin-independent mechanisms contribute to AI, which are likely mediated by a direct effect of inflammatory cytokines on erythropoiesis. In this study, we used wild-type, hepcidin knockout (Hamp-KO) and interleukin 6 knockout (IL-6-KO) mice as models of AI. AI was induced with heat-killed Brucella abortus (BA). The distinct roles of iron metabolism and inflammation triggered by interleukin 6 and hepcidin were investigated. BA-treated wild-type mice showed increased expression of hepcidin and inflammatory cytokines, as well as transitory suppression of erythropoiesis and shortened red blood cell lifespan, all of which contributed to the severe anemia of these mice. In contrast, BA-treated Hamp-KO or IL-6-KO mice showed milder anemia and faster recovery compared with normal mice. Moreover, they exhibited different patterns in the development and resolution of anemia, supporting the notion that interleukin 6 and hepcidin play distinct roles in modulating erythropoiesis in AI.

Macrophages support pathological erythropoiesis in polycythemia vera and ß-thalassemia.

Regulation of erythropoiesis is achieved by the integration of distinct signals. Among them, macrophages are emerging as erythropoietin-complementary regulators of erythroid development, particularly under stress conditions. We investigated the contribution of macrophages to physiological and pathological conditions of enhanced erythropoiesis. We used mouse models of induced anemia, polycythemia vera and ß-thalassemia in which macrophages were chemically depleted. Our data indicate that macrophages contribute decisively to recovery from induced anemia, as well as the pathological progression of polycythemia vera and ß-thalassemia, by modulating erythroid proliferation and differentiation. We validated these observations in primary human cultures, showing a direct impact of macrophages on the proliferation and enucleation of erythroblasts from healthy individuals and patients with polycythemia vera or ß-thalassemia. The contribution of macrophages to stress and pathological erythropoiesis, which we have termed stress erythropoiesis macrophage-supporting activity, may have therapeutic implications.

Combined treatment with recombinant tissue plasminogen activator and dexamethasone phosphate-containing liposomes improves neurological outcome and restricts lesion progression after embolic stroke in rats.

Variable efficacies have been reported for glucocorticoid drugs as anti-inflammatory treatment after stroke. We applied an alternative drug delivery strategy, by injection of dexamethasone phosphate-containing liposomes in combination with recombinant tissue plasminogen activator (rtPA), in an experimental stroke model, and tested the hypothesis that this approach improves behavioral recovery and reduces lesion growth. Rats were subjected to right middle cerebral artery occlusion with a blood clot. After 2 h, animals were intravenously injected with rtPA plus empty long-circulating liposomes (LCL), free dexamethasone phosphate (DXP), or DXP-containing LCL (LCL-DXP). Neurological status was evaluated with different behavioral tests up to 7 days after stroke. Lesion development was assessed by magnetic resonance imaging of tissue and perfusion parameters from 0-2 h until 7 days after stroke. Expression of brain inflammatory markers was measured with RT-PCR at post-stroke day 7. Treatment with rtPA plus LCL-DXP resulted in significantly improved behavioral outcome as compared to treatment with rtPA plus empty LCL or free DXP. Acute and final brain lesion sizes were comparable between treatment groups; however a predictive algorithm revealed a significantly larger salvaged tissue area after treatment with LCL-DXP. We conclude that delivery of dexamethasone phosphate via LCL in combination with rtPA-induced thrombolysis can significantly improve outcome after stroke. Furthermore, magnetic resonance imaging-based predictive algorithms provide a sensitive means to measure treatment effects on lesion development.

Screening of budesonide nanoformulations for treatment of inflammatory bowel disease in an inflamed 3D cell-culture model.

Drug formulation screenings for treatment of inflammatory bowel disease (IBD) are mostly conducted in chemically induced rodent models that represent acute injury-caused inflammation instead of a chronic condition. To accurately screen drug formulations for chronic IBD, a relevant model that mimics the chronic condition in vitro is urgently needed. In an effort to reduce and potentially replace this scientifically and ethically questionable animal testing for IBD drugs, our laboratory has developed an in vitro model for the inflamed intestinal mucosa observed in chronic IBD, which allows high-throughput screening of anti-inflammatory drugs and their formulations. The in vitro model consists of intestinal epithelial cells, human blood-derived macrophages, and dendritic cells that are stimulated by the inflammatory cytokine interleukin-1ß. In this study, the model was utilized for evaluation of the efficacy and deposition of budesonide, an anti-inflammatory drug, in three different pharmaceutical formulations: (1) a free drug solution, (2) encapsulated into PLGA nanoparticles, and (3) encapsulated into liposomes. The in vitro model of the inflamed intestinal mucosa demonstrated its ability to differentiate therapeutic efficacy among the formulations while maintaining the convenience of conventional in vitro studies and adequately representing the complex pathophysiological changes observed in vivo.

Glucocorticoid-loaded core-cross-linked polymeric micelles with tailorable release kinetics for targeted therapy of rheumatoid arthritis.

Polymerizable and hydrolytically cleavable dexamethasone (DEX, red dot in picture) derivatives were covalently entrapped in core-cross-linked polymeric micelles that were prepared from a thermosensitive block copolymer (yellow and gray building block). By varying the oxidation degree of the thioether in the drug linker, the release rate of DEX could be controlled. The DEX-loaded micelles were used for efficient treatment of inflammatory arthritis in two animal models.

Drug targeting systems for inflammatory disease: one for all, all for one.

In various systemic disorders, structural changes in the microenvironment of diseased tissues enable both passive and active targeting of therapeutic agents to these tissues. This has led to a number of targeting approaches that enhance the accumulation of drugs in the target tissues, making drug targeting an attractive strategy for the treatment of various diseases. Remarkably, the strategic principles that form the basis of drug targeting are often employed for tumor targeting, while chronic inflammatory diseases appear to draw much less attention. To provide the reader with a general overview of the current status of drug targeting to inflammatory diseases, the passive and active targeting strategies that have been used for the treatment of rheumatoid arthritis (RA) and multiple sclerosis (MS) are discussed. The last part of this review addresses the dualism of platform technology-oriented ("one for all") and disease-oriented drug targeting research ("all for one"), both of which are key elements of effective drug targeting research.

Liposomes as carriers for colchicine-derived prodrugs: vascular disrupting nanomedicines with tailorable drug release kinetics.

Newly formed tumor vasculature has proven to be an effective target for tumor therapy. A strategy to attack this angiogenic tumor vasculature is to initiate local blood vessel congestion and consequently induce massive tumor cell necrosis. Vascular disrupting agents (VDAs) typically bind to tubulin and consequently disrupt microtubule dynamics. Colchicine and its derivatives (colchicinoids) are very potent tubulin binding compounds but have a narrow therapeutic index, which may be improved by employing a liposomal targeting strategy. However, as a result of their physicochemical properties, colchicinoids are problematic to retain in liposomes, as they are released relatively rapidly upon encapsulation. To overcome this limitation, two hydrolyzable PEGylated derivatives of colchicine were developed for encapsulation into the aqueous core of long-circulating liposomes: a moderately rapid hydrolyzing PEGylated colchicinoid containing a glycolic acid linker (prodrug I), and a slower hydrolyzing PEGylated colchicinoid with a lactic acid linker (prodrug II). Hydrolysis studies at 37°C and pH 7.4 showed that prodrug I possessed relatively rapid conversion characteristics (t(1/2)=5.4 h) whereas prodrug II hydrolyzed much slower (t(1/2)=217 h). Upon encapsulation into liposomes, colchicine was released rapidly, whereas both PEGylated colchicine derivatives were efficiently retained and appeared to be released only after cleavage of the PEG-linker. This study therefore demonstrates that, in contrast to colchicine, these novel PEGylated colchicine-derived prodrugs are retained within the aqueous interior after encapsulation into liposomes, and that the release of the active parent can be controlled by using different biodegradable linkers.

MRI evaluation of the antitumor activity of paramagnetic liposomes loaded with prednisolone phosphate.

The design of long circulating liposomes co-loaded with the glucocorticoid prednisolone phosphate (PLP) and the amphiphilic paramagnetic contrast agent Gd-DOTAMA(C(18))(2) allowed the MRI-guided in vivo visualization of the delivery and biodistribution of PLP, as well as the monitoring of drug efficacy. The performance of this theranostic probe was investigated in a mouse model bearing a melanoma B16 syngeneic tumor. The release kinetics of the drug were evaluated in vitro where it displayed a peculiar behavior characterized by a fast process (completed in few hours) involving only a small portion (<5%) of the drug. Interestingly, the incorporation of the amphiphilic imaging reporter in the liposomal bilayer slightly increased the amount of the fast-release portion (<10%), thus suggesting that it could be attributed to a drug fraction embedded in the liposomal bilayer. In fact, the release of a hydrophilic imaging probe encapsulated in the inner core of the same long circulating liposomes formulated for carrying the drug, displayed different, single-step, kinetics. The in vivo monitoring of the antitumor activity of the nanomedicine revealed that the incorporation of the MRI probe into the liposome bilayer did not significantly affect the drug efficacy. The in vivo experiments also indicated a relevant and fast liposome uptake from macrophage-rich organs like spleen and liver, which reduced the tumor accumulation of the liposomes. The accumulation of the amphipatic MRI label caused the occurrence of a long-term residual T(1) contrast still detectable 1week after injection.

Critical factors in the development of tumor-targeted anti-inflammatory nanomedicines.

Tumors are complex and dynamic assemblies of different cell types within an atypical/abnormal architecture. Tumor-associated macrophages, mast cells, dendritic cells, eosinophils, fibroblasts are present in and around tumors and contribute to an abundant, deregulated and long lasting chronic inflammation, a permissive microenvironment for tumor proliferation. The fact that tumor-initiated inflammation overrides protective immunity and supports tumor development, combined with the successful strategy of liposomal drug targeting to tumor tissues, gave rise to a novel attractive antitumor therapeutic strategy: targeting anti-inflammatory drugs to tumors. As many natural bioactive compounds have anti-inflammatory properties and act by targeting multiple signaling pathways, their medicinal application involves the treatment of cancer. The nanoformulation into liposomes circumvents solubility, stability, bioavailability issues associated with these compounds, while promoting tumor drug accumulation. Liposomal formulations of resveratrol, carvacrol phosphate, pterostilbene phosphate, N-(3-oxo-dodecanoyl)-l-Homoserine lactone, caffeic acid and caffeic acid phenethyl ester (CAPE) were prepared and characterized. In vivo, B16F10 melanoma bearing mice seem not to be responsive to liposomal treatments of natural compounds. The specific combination of tumor model, drug delivery system and drug potency are crucial factors that should be considered in the development and evaluation of new nanomedicines for cancer-inflammation therapy, especially when translating from preclinical models.

A polymeric colchicinoid prodrug with reduced toxicity and improved efficacy for vascular disruption in cancer therapy.

Colchicinoids are very potent tubulin-binding compounds, which interfere with microtubule formation, giving them strong cytotoxic properties, such as cell mitosis inhibition and induction of microcytoskeleton depolymerization. While this makes them promising vascular disrupting agents (VDAs) in cancer therapy, their dose-limiting toxicity has prevented any clinical application for this purpose. Therefore, colchicinoids are considered attractive lead molecules for the development of novel vascular disrupting nanomedicine. In a previous study, a polymeric colchicinoid prodrug that showed favorable hydrolysis characteristics at physiological conditions was developed. In the current study, this polymeric colchicinoid prodrug was evaluated in vitro and in vivo for its toxicity and vascular disrupting potential. Cell viability studies with human umbilical vein endothelial cells, as an in vitro measure for colchicine activity, reflected the degradation kinetics of the prodrug accordingly. Upon intravenous treatment, in vivo, of B16F10 melanoma-bearing mice with colchicine or with the polymeric colchicinoid prodrug, apparent vascular disruption and consequent tumor necrosis was observed for the prodrug but not for free colchicine at an equivalent dose. Moreover, a five-times-higher dose of the prodrug was well tolerated, indicating reduced toxicity. These findings demonstrate that the polymeric colchicinoid prodrug has a substantially improved efficacy/toxicity ratio compared with that of colchicine, making it a promising VDA for cancer therapy.

A biodegradable antibiotic delivery system based on poly-(trimethylene carbonate) for the treatment of osteomyelitis.

BACKGROUND AND PURPOSE: Many investigations on biodegradable materials acting as an antibiotic carrier for local drug delivery are based on poly(lactide). However, the use of poly(lactide) implants in bone has been disputed because of poor bone regeneration at the site of implantation. Poly(trimethylene carbonate) (PTMC) is an enzymatically degradable polymer that does not produce acidic degradation products. We explored the suitability of PTMC as an antibiotic releasing polymer for the local treatment of osteomyelitis. METHODS: This study addressed 2 separate attributes of PTMC: (1) the release kinetics of gentamicin-loaded PTMC and (2) its behavior in inhibiting biofilm formation. Both of these characteristics were compared with those of commercially available gentamicin-loaded poly(methylmethacrylate) (PMMA) beads, which are commonly used in the local treatment of osteomyelitis. RESULTS: In a lipase solution that mimics the in vivo situation, PTMC discs with gentamicin incorporated were degraded by surface erosion and released 60% of the gentamicin within 14 days. This is similar to the gentamicin release from clinically used PMMA beads. Moreover, biofilm formation by Staphylococcus aureus was inhibited by approximately 80% over at least 14 days in the presence of gentamicin-loaded PTMC discs. This is similar to the effect of gentamicin-loaded PMMA beads. In the absence of the lipase, surface erosion of PTMC discs did not occur and gentamicin release and biofilm inhibition were limited. INTERPRETATION: Since gentamicin-loaded PTMC discs show antibiotic release characteristics and biofilm inhibition characteristics similar to those of gentamicin-loaded PMMA beads, PTMC appears to be a promising biodegradable carrier in the local treatment of osteomyelitis.