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Maaike VJ Braham,1 Anil K Deshantri,2,3 Monique C Minnema,4 F Cumhur Öner,1 Raymond M Schiffelers,2 Marcel HAM Fens,2,5 Jacqueline Alblas1 1Department of Orthopaedics, University Medical Center Utrecht, Utrecht, the Netherlands; 2Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, the Netherlands; 3Department of Pharmacology, Sun Pharma Advanced Research Company Limited, Vadodara, Gujarat, India; 4Department of Hematology, University Medical Center Utrecht Cancer Center, Utrecht, the Netherlands; 5Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, the Netherlands Purpose: Liposomal drug delivery can improve the therapeutic index of treatments for multiple myeloma. However, an appropriate 3D model for the in vitro evaluation of liposomal drug delivery is lacking. In this study, we applied a previously developed 3D bone marrow (BM) myeloma model to examine liposomal drug therapy. Material and methods: Liposomes of different sizes (~75–200 nm) were tested in a 3D BM myeloma model, based on multipotent mesenchymal stromal cells, endothelial progenitor cells, and myeloma cells cocultured in hydrogel. The behavior and efficacy of liposomal drug therapy was investigated, evaluating the feasibility of testing liposomal drug delivery in 3D in vitro. Intracellular uptake of untargeted and integrin α4β1 (very late antigen-4) targeted liposomes was compared in myeloma and supporting cells, as well as the effectivity of free and liposome-encapsulated chemotherapy (bortezomib, doxorubicin). Either cocultured myeloma cell lines or primary CD138+ myeloma cells received the treatments. Results: Liposomes (~75–110 nm) passively diffused throughout the heterogeneously porous (~80–850 nm) 3D hydrogel model after insertion. Cellular uptake of liposomes was observed and was increased by targeting very late antigen-4. Liposomal bortezomib and doxorubicin showed increased cytotoxic effects toward myeloma cells compared with the free drugs, using either a cell line or primary myeloma cells. Cytotoxicity toward supporting BM cells was reduced using liposomes. Conclusion: The 3D model allows the study of liposome-encapsulated molecules on multiple myeloma and supporting BM cells, looking at cellular targeting, and general efficacy of the given therapy. The advantages of liposomal drug delivery were demonstrated in a primary myeloma model, enabling the study of patient-to-patient responses to potential drugs and treatment regimes. Keywords: liposomes, targeted delivery, tumor microenvironment, drug sensitivity and resistance testing, multiple myeloma

Bart J Crielaard1, Steffen van der Wal1, Twan Lammers2, Huong Thu Le1, Wim E Hennink1, Raymond M Schiffelers1, Gert Storm1, Marcel HAM Fens11Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands; 2Department of Experimental Molecular Imaging, RWTH Aachen University, Aachen, Germany The first two authors contributed equally to this work. Abstract: 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. Keywords: colchicine, prodrug, nanomedicines, cancer, vascular disrupting agents

Heart failure is a serious condition that results from the extensive loss of specialized cardiac muscle cells called cardiomyocytes (CMs), typically caused by myocardial infarction (MI). Messenger RNA (mRNA) therapeutics are emerging as a very promising gene medicine for regenerative cardiac therapy. To date, lipid nanoparticles (LNPs) represent the most clinically advanced mRNA delivery platform. Yet, their delivery efficiency has been limited by their endosomal entrapment after endocytosis. Previously, we demonstrated that a pair of complementary coiled-coil peptides (CPE4/CPK4) triggered efficient fusion between liposomes and cells, bypassing endosomal entrapment and resulting in efficient drug delivery. Here, we modified mRNA-LNPs with the fusogenic coiled-coil peptides and demonstrated efficient mRNA delivery to difficult-to-transfect induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs). As proof of in vivo applicability of these fusogenic LNPs, local administration via intramyocardial injection led to significantly enhanced mRNA delivery and concomitant protein expression. This represents the successful application of the fusogenic coiled-coil peptides to improve mRNA-LNPs transfection in the heart and provides the potential for the advanced development of effective regenerative therapies for heart failure.

To improve the reliability of in vitro release studies of drug delivery systems, we developed a novel in vitro method for the evaluation of drug release from polymeric micelles in complex biological media. Polymeric micelles based on poly(N-2-hydroxypropyl methacrylamide)-block-poly(N-2-benzoyloxypropyl methacrylamide) (p(HPMAm)-b-p(HPMAm-Bz)) of which 10% of the chains was functionalized with biotin at the p(HPMAm) terminus were prepared using a solvent extraction method. The size of the micelles when loaded with a hydrophobic agent, namely paclitaxel (a clinically used cytostatic drug) or curcumin (a compound with multiple pharmacological activities), was around 65 nm. The biotin decoration allowed the binding of the micelles to streptavidin-coated magnetic beads which occurred within 10 min and reached a binding efficiency of 90 ± 6%. Drug release in different media was studied after the magnetic separation of micelles bound to the streptavidin-coated beads, by determination of the released drug in the media as well as the retained drug in the micellar fraction bound to the beads. The in vitro release of paclitaxel and curcumin at 37 °C in PBS, PBS containing 2% v/v Tween 80, PBS containing 4.5% w/v bovine serum albumin, mouse plasma, and whole mouse blood was highly medium-dependent. In all media studied, paclitaxel showed superior micellar retention compared to curcumin. Importantly, the presence of serum proteins accelerated the release of both paclitaxel and curcumin. The results presented in this study show great potential for predicting drug release from nanomedicines in biological media which in turn is crucial for their further pharmaceutical development., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Aim: A photosensitizer (PS) delivery and comprehensive tumor targeting platform was developed that is centered on the photosensitization of key pharmacological targets in solid tumors (cancer cells, tumor vascular endothelium, and cellular and non-cellular components of the tumor microenvironment) before photodynamic therapy (PDT). Interstitially targeted liposomes (ITLs) encapsulating zinc phthalocyanine (ZnPC) and aluminum phthalocyanine (AlPC) were formulated for passive targeting of the tumor microenvironment. In previous work it was established that the PEGylated ITLs were taken up by cultured cholangiocarcinoma cells. The aim of this study was to verify previous results in cancer cells and to determine whether the ITLs can also be used to photosensitize cells in the tumor microenvironment and vasculature. Following positive results, rudimentary in vitro and in vivo experiments were performed with ZnPC-ITLs and AlPC-ITLs as well as their water-soluble tetrasulfonated derivatives (ZnPCS4 and AlPCS4) to assemble a research dossier and bring this platform closer to clinical transition., Methods: Flow cytometry and confocal microscopy were employed to determine ITL uptake and PS distribution in cholangiocarcinoma (SK-ChA-1) cells, endothelial cells (HUVECs), fibroblasts (NIH-3T3), and macrophages (RAW 264.7). Uptake of ITLs by endothelial cells was verified under flow conditions in a flow chamber. Dark toxicity and PDT efficacy were determined by cell viability assays, while the mode of cell death and cell cycle arrest were assayed by flow cytometry. In vivo systemic toxicity was assessed in zebrafish and chicken embryos, whereas skin phototoxicity was determined in BALB/c nude mice. A PDT efficacy pilot was conducted in BALB/c nude mice bearing human triple-negative breast cancer (MDA-MB-231) xenografts., Results: The key findings were that (1) photodynamically active PSs (i.e., all except ZnPCS4) were able to effectively photosensitize cancer cells and non-cancerous cells; (2) following PDT, photodynamically active PSs were highly toxic-to-potent as per anti-cancer compound classification; (3) the photodynamically active PSs did not elicit notable systemic toxicity in zebrafish and chicken embryos; (4) ITL-delivered ZnPC and ZnPCS4 were associated with skin phototoxicity, while the aluminum-containing PSs did not exert detectable skin phototoxicity; and (5) ITL-delivered ZnPC and AlPC were equally effective in their tumor-killing capacity in human tumor breast cancer xenografts and superior to other non-phthalocyanine PSs when appraised on a per mole administered dose basis., Conclusions: AlPC(S4) are the safest and most effective PSs to integrate into the comprehensive tumor targeting and PS delivery platform. Pending further in vivo validation, these third-generation PSs may be used for multi-compartmental tumor photosensitization., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The therapeutic potential of antigen-specific regulatory T cells (Treg) has been extensively explored, leading to the development of several tolerogenic vaccines. Dexamethasone-antigen conjugates represent a prominent class of tolerogenic vaccines that enable coordinated delivery of antigen and dexamethasone to target immune cells. The importance of nonspecific albumin association towards the biodistribution of antigen-adjuvant conjugates has gained increasing attention, by which hydrophobic and electrostatic interactions govern the association capacity. Using an ensemble of computational and experimental techniques, we evaluate the impact of charged residues adjacent to the drug conjugation site in dexamethasone-antigen conjugates (Dex-K/E4-OVA323, K: lysine, E: glutamate) towards their albumin association capacity and induction of antigen-specific Treg. We find that Dex-K4-OVA323 possesses a higher albumin association capacity than Dex-E4-OVA323, leading to enhanced liver distribution and antigen-presenting cell uptake. Furthermore, using an OVA323-specific adoptive-transfer mouse model, we show that Dex-K4-OVA323 selectively upregulated OVA323-specific Treg cells, whereas Dex-E4-OVA323 exerted no significant effect on Treg cells. Our findings serve as a guide to optimize the functionality of dexamethasone-antigen conjugate amid switching vaccine epitope sequences. Moreover, our study demonstrates that moderating the residues adjacent to the conjugation sites can serve as an engineering approach for future peptide-drug conjugate development., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Induction of antigen-specific immune tolerance has emerged as the next frontier in treating autoimmune disorders, including atherosclerosis and graft-vs-host reactions during transplantation. Nanostructures are under investigation as a platform for the coordinated delivery of critical components, i.e., the antigen epitope combined with tolerogenic agents, to the target immune cells and subsequently induce tolerance. In the present study, the utility of supramolecular peptide nanofibers to induce antigen-specific immune tolerance was explored. To study the influence of surface charges of the nanofibers towards the extent of the induced immune response, the flanking charge residues at both ends of the amphipathic fibrillization peptide sequences were varied. Dexamethasone, an immunosuppressive glucocorticoid drug, and the ovalbumin-derived OVA323-339 peptide that binds to I-A(d) MHC Class II were covalently linked at either end of the peptide sequences. It was shown that the functional extensions did not alter the structural integrity of the supramolecular nanofibers. Furthermore, the surface charges of the nanofibers were modulated by the inclusion of charged residues. Dendritic cell culture assays suggested that nanofiber of less negative ζ-potential can augment the antigen-specific tolerogenic response. Our findings illustrate a molecular approach to calibrate the tolerogenic response induced by peptide nanofibers, which pave the way for better design of future tolerogenic immunotherapies., Competing Interests: Declaration of Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

PEGylation of lipid-based nanoparticles and other nanocarriers is widely used to increase their stability and plasma half-life. However, either pre-existing or de novo formed anti-PEG antibodies can induce hypersensitivity reactions and accelerated blood clearance through binding to the nanoparticle surfaces, leading to activation of the complement system. In this study, we investigated the consequences and mechanisms of complement activation by anti-PEG antibodies interacting with different types of PEGylated lipid-based nanoparticles. By using both liposomes loaded with different (model) drugs and LNPs loaded with mRNA, we demonstrate that complement activation triggered by anti-PEG antibodies can compromise the bilayer/surface integrity, leading to premature drug release or exposure of their mRNA contents to serum proteins. Anti-PEG antibodies also can induce deposition of complement fragments onto the surface of PEGylated lipid-based nanoparticles and induce the release of fluid phase complement activation products. The role of the different complement pathways activated by lipid-based nanoparticles was studied using deficient sera and/or inhibitory antibodies. We identified a major role for the classical complement pathway in the early activation events leading to the activation of C3. Our data also confirm the essential role of amplification of C3 activation by alternative pathway components in the lysis of liposomes. Finally, the levels of pre-existing anti-PEG IgM antibodies in plasma of healthy donors correlated with the degree of complement activation (fixation and lysis) induced upon exposure to PEGylated liposomes and mRNA-LNPs. Taken together, anti-PEG antibodies trigger complement activation by PEGylated lipid-based nanoparticles, which can potentially compromise their integrity, leading to premature drug release or cargo exposure to serum proteins., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Serine proteases are essential for many physiological processes and require tight regulation by serine protease inhibitors (SERPINs). A disturbed SERPIN-protease balance may result in disease. The reactive center loop (RCL) contains an enzymatic cleavage site between the P1 through P1' residues that controls SERPIN specificity. This RCL can be modified to improve SERPIN function; however, a lack of insight into sequence-function relationships limits SERPIN development. This is complicated by more than 25 billion mutants needed to screen the entire P4 to P4' region. Here, we developed a platform to predict the effects of RCL mutagenesis by using α1-antitrypsin as a model SERPIN. We generated variants for each of the residues in P4 to P4' region, mutating them into each of the 20 naturally occurring amino acids. Subsequently, we profiled the reactivity of the resulting 160 variants against seven proteases involved in coagulation. These profiles formed the basis of an in silico prediction platform for SERPIN inhibitory behavior with combined P4 to P4' RCL mutations, which were validated experimentally. This prediction platform accurately predicted SERPIN behavior against five out of the seven screened proteases, one of which was activated protein C (APC). Using these findings, a next-generation APC-inhibiting α1-antitrypsin variant was designed (KMPR/RIRA; / indicates the cleavage site). This variant attenuates blood loss in an in vivo hemophilia A model at a lower dosage than the previously developed variant AIKR/KIPP because of improved potency and specificity. We propose that this SERPIN-based RCL mutagenesis approach improves our understanding of SERPIN behavior and will facilitate the design of therapeutic SERPINs., Competing Interests: The authors declare no competing interest.

Curcumin-loaded polymeric micelles composed of poly(ethylene glycol)- b -poly( N -2-benzoyloxypropyl methacrylamide) (mPEG- b -p(HPMA-Bz)) were prepared to solubilize and improve the pharmacokinetics of curcumin. Curcumin-loaded micelles were prepared by a nanoprecipitation method using mPEG 5kDa - b -p(HPMA-Bz) copolymers with varying molecular weight of the hydrophobic block (5.2, 10.0, and 17.1 kDa). At equal curcumin loading, micelles composed of mPEG 5kDa - b -p(HPMA-Bz) 17.1kDa showed better curcumin retention in both phosphate-buffered saline (PBS) and plasma at 37 °C than micelles based on block copolymers with smaller hydrophobic blocks. No change in micelle size was observed during 24 h incubation in plasma using asymmetrical flow field-flow fractionation (AF 4 ), attesting to particle stability. However, 22-49% of the curcumin loading was released from the micelles during 24 h from formulations with the highest to the lowest molecular weight p(HPMA-Bz), respectively, in plasma. AF 4 analysis further showed that the released curcumin was subsequently solubilized by albumin. In vitro analyses revealed that the curcumin-loaded mPEG 5kDa - b -p(HPMA-Bz) 17.1kDa micelles were internalized by different types of cancer cells, resulting in curcumin-induced cell death. Intravenously administered curcumin-loaded, Cy7-labeled mPEG 5kDa - b -p(HPMA-Bz) 17.1kDa micelles in mice at 50 mg curcumin/kg showed a long circulation half-life for the micelles ( t 1/2 = 42 h), in line with the AF 4 results. In contrast, the circulation time of curcumin was considerably shorter than that of the micelles ( t 1/2α = 0.11, t 1/2β = 2.5 h) but ∼5 times longer than has been reported for free curcumin ( t 1/2α = 0.02 h). The faster clearance of curcumin in vivo compared to in vitro studies can be attributed to the interaction of curcumin with blood cells. Despite the excellent solubilizing effect of these micelles, no cytostatic effect was achieved in neuroblastoma-bearing mice, possibly because of the low sensitivity of the Neuro2A cells to curcumin.

Glucocorticoids are the cornerstone in the clinic for treatment of hematological malignancies, including multiple myeloma. Nevertheless, poor pharmacokinetic properties of glucocorticoids require high and frequent dosing with the off-target adverse effects defining the maximum dose. Recently, nanomedicine formulations of glucocorticoids have been developed that improve the pharmacokinetic profile, limit adverse effects and improve solid tumor accumulation. Multiple myeloma is a hematological malignancy characterized by uncontrolled growth of plasma cells. These tumors initiate increased angiogenesis and microvessel density in the bone marrow, which might be exploited using nanomedicines, such as liposomes. Nano-sized particles can accumulate as a result of the increased vascular leakiness at the bone marrow tumor lesions. Pre-clinical screening of novel anti-myeloma therapeutics in vivo requires a suitable animal model that represents key features of the disease. In this study, we show that fluorescently labeled long circulating liposomes were found in plasma up to 24 h after injection in an advanced human-mouse hybrid model of multiple myeloma. Besides the organs involved in clearance, liposomes were also found to accumulate in tumor bearing human-bone scaffolds. The therapeutic efficacy of liposomal dexamethasone phosphate was evaluated in this model showing strong tumor growth inhibition while free drug being ineffective at an equivalent dose (4 mg/kg) regimen. The liposomal formulation slightly reduced total body weight of myeloma-bearing mice during the course of treatment, which appeared reversible when treatment was stopped. Liposomal dexamethasone could be further developed as monotherapy or could fit in with existing therapy regimens to improve therapeutic outcomes for multiple myeloma., (Copyright © 2019. Published by Elsevier B.V.)

Polyphosphate is an inorganic polymer that can potentiate several interactions in the blood coagulation system. Blood platelets contain polyphosphate, and the secretion of platelet-derived polyphosphate has been associated with increased thrombus formation and activation of coagulation factor XII. However, the small polymer size of secreted platelet polyphosphate limits its capacity to activate factor XII in vitro. Thus, the mechanism by which platelet polyphosphate contributes to thrombus formation remains unclear. Using live-cell imaging, confocal and electron microscopy, we show that activated platelets retain polyphosphate on their cell surface. The apparent polymer size of membrane-associated polyphosphate largely exceeds that of secreted polyphosphate. Ultracentrifugation fractionation experiments revealed that membrane-associated platelet polyphosphate is condensed into insoluble spherical nanoparticles with divalent metal ions. In contrast to soluble polyphosphate, membrane-associated polyphosphate nanoparticles potently activate factor XII. Our findings identify membrane-associated polyphosphate in a nanoparticle state on the surface of activated platelets. We propose that these polyphosphate nanoparticles mechanistically link the procoagulant activity of platelets with the activation of coagulation factor XII., (© 2017 by The American Society of Hematology.)

Cancers are abundantly infiltrated by inflammatory cells that are modulated by tumor cells to secrete mediators fostering tumor cell survival and proliferation. Therefore, agents that interfere with inflammatory signaling molecules or specific immune cell populations have been investigated as anticancer drugs. Corticosteroids are highly potent anti-inflammatory drugs, whose activity is intensified when targeted by nanocarrier systems. Liposome-targeted corticosteroids have been shown to inhibit tumor growth in different syngeneic murine tumor models as well as human xenograft mouse models, which is attributed to a switch in the tumor microenvironment from a pro-inflammatory to an anti-inflammatory state. Despite the recognized value of implantation tumor models in preclinical research, the "acute" inflammation induced by inoculation of tumor cells together with the exponential tumor growth in a relatively short period of time does not resemble slow progressive human disease that develops in situ. Therefore, in this study, the antitumor effect of liposomal corticosteroids was investigated in a clinically more relevant setting of transgenic mice developing spontaneous breast carcinomas. Here we show that liposomal prednisolone phosphate inhibits the growth of spontaneous breast carcinoma. Interestingly, the liposomal prednisolone was significantly more active than free drug. At 72h after injection of the liposomal formulation, 3μg prednisolone per gram of tumor tissue was recovered whereas no drug could be recovered after injection of the free agent. This indicates that, despite etiological and morphological differences between implanted and spontaneous tumor models, EPR-mediated accumulation of drug occurs to similar extent in this spontaneous mammary carcinoma model as in the syngeneic tumor models. Finally, we analyzed miRNA profiles in the MMTV/neu model and showed that the top 10 of miRNAs in the MMTV/neu tumor consisted of miRNAs with a known involvement in breast carcinoma proliferation and metastasis. The only exception was the appearance of miR-146b, a known inflammation-regulating miRNA species, after liposomal prednisolone treatment., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This study reveals a novel interaction between deoxyhemoglobin, nitrite and the non-toxic compound, RRx-001, to generate supraphysiologic levels of nitric oxide (NO) in blood. We characterize the nitrite reductase activity of deoxyhemoglobin, which in the presence of bound RRx-001 reduces nitrite at a much faster rate, leading to markedly increased NO generation. These data expand on the paradigm that hemoglobin generates NO via nitrite reduction during hypoxia and ischemia when nitric oxide synthase (NOS) function is limited. Here, we demonstrate that RRx-001 greatly enhances NO generation from nitrite reduction. RRx-001 is thus the first example of a functional superagonist for nitrite reductase. We hypothesize that physiologically this reaction releases the potentially cytotoxic effector NO selectively in hypoxic tumor regions. It may be that a binary NO-H2O2 trigger is indirectly responsible for the observed tumoricidal activity of RRx-001 since NO is known to inhibit mitochondrial respiration.

Treatment of cancer patients with taxane-based chemotherapeutics, such as paclitaxel (PTX), is complicated by their narrow therapeutic index. Polymeric micelles are attractive nanocarriers for tumor-targeted delivery of PTX, as they can be tailored to encapsulate large amounts of hydrophobic drugs and achiv prolonged circulation kinetics. As a result, PTX deposition in tumors is increased, while drug exposure to healthy tissues is reduced. However, many PTX-loaded micelle formulations suffer from low stability and fast drug release in the circulation, limiting their suitability for systemic drug targeting. To overcome these limitations, we have developed PTX-loaded micelles which are stable without chemical cross-linking and covalent drug attachment. These micelles are characterized by excellent loading capacity and strong drug retention, attributed to π-π stacking interaction between PTX and the aromatic groups of the polymer chains in the micellar core. The micelles are based on methoxy poly(ethylene glycol)-b-(N-(2-benzoyloxypropyl)methacrylamide) (mPEG-b-p(HPMAm-Bz)) block copolymers, which improved the pharmacokinetics and the biodistribution of PTX, and substantially increased PTX tumor accumulation (by more than 2000%; as compared to Taxol or control micellar formulations). Improved biodistribution and tumor accumulation were confirmed by hybrid μCT-FMT imaging using near-infrared labeled micelles and payload. The PTX-loaded micelles were well tolerated at different doses, while they induced complete tumor regression in two different xenograft models (i.e., A431 and MDA-MB-468). Our findings consequently indicate that π-π stacking-stabilized polymeric micelles are promising carriers to improve the delivery of highly hydrophobic drugs to tumors and to increase their therapeutic index.

Extracellular vesicles (EVs) are membrane-derived particles surrounded by a (phospho)lipid bilayer that are released by cells in the human body. In addition to direct cell-to-cell contact and the secretion of soluble factors, EVs function as another mechanism of intercellular communication. These vesicles are able to efficiently deliver their parental cell-derived molecular cargo to recipient cells, which can result in structural changes at an RNA, protein, or even phenotypic level. For this reason, EVs have recently gained much interest for drug delivery purposes. In contrast to these 'natural delivery systems', synthetic (phospho)lipid vesicles, or liposomes, have been employed as drug carriers for decades, resulting in several approved liposomal nanomedicines used in the clinic. This review discusses the similarities and differences between EVs and liposomes with the focus on features that are relevant for drug delivery purposes such as circulation time, biodistribution, cellular interactions and cargo loading. By applying beneficial features of EVs to liposomes and vice versa, improved drug carriers can be developed which will advance the field of nanomedicines and ultimately improve patient outcomes. While the application of EVs for therapeutic drug delivery is still in its infancy, issues regarding the understanding of EV biogenesis, large-scale production and in vivo interactions need to be addressed in order to develop successful and cost-effective EV-based drug delivery systems., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Nitric oxide (NO) is a key regulator of vascular tone. Endothelial nitric oxide synthase (eNOS) is responsible for NO generation under normoxic conditions. Under hypoxia however, eNOS is inactive and red blood cells (RBC) provide an alternative NO generation pathway from nitrite to regulate hypoxic vasodilation. While nitrite reductase activity of hemoglobin is well acknowledged, little is known about generation of NO by intact RBC with physiological hemoglobin concentrations. We aimed to develop and apply a new approach to provide insights in the ability of RBC to convert nitrite into NO under hypoxic conditions. We established a novel experimental setup to evaluate nitrite uptake and the release of NO from RBC into the gas-phase under different conditions. NO measurements were similar to well-established clinical measurements of exhaled NO. Nitrite uptake was rapid, and after an initial lag phase NO release from RBC was constant in time under hypoxic conditions. The presence of oxygen greatly reduced NO release, whereas inhibition of eNOS and xanthine oxidoreductase (XOR) did not affect NO release. A decreased pH increased NO release under hypoxic conditions. Hypothermia lowered NO release, while hyperthermia increased NO release. Whereas fetal hemoglobin did not alter NO release compared to adult hemoglobin, sickle RBC showed an increased ability to release NO. Under all conditions nitrite uptake by RBC was similar. This study shows that nitrite uptake into RBC is rapid and release of NO into the gas-phase continues for prolonged periods of time under hypoxic conditions. Changes in the RBC environment such as pH, temperature or hemoglobin type, affect NO release.

Inflammation is an important component of various cancers and its inflammatory cells and mediators have been shown to have prognostic potential. Tumor-infiltrating mast cells can promote tumor growth and angiogenesis, but the mechanism of mast cell activation is unclear. In earlier studies, we demonstrated that immunoglobulin free light chains (FLC) can trigger mast cells in an antigen-specific manner. Increased expression of FLC was observed within stroma of various human cancers including those of breast, colon, lung, pancreas, kidney and skin, and FLC expression co-localized with areas of mast cell infiltration. In a large cohort of breast cancer patients, FLC expression was shown associated with basal-like cancers with an aggressive phenotype. Moreover, lambda FLC was found expressed in areas of inflammatory infiltration and its expression was significantly associated with poor clinical outcome. Functional importance of FLCs was shown in a murine B16F10 melanoma model, where inhibition of FLC-mediated mast cell activation strongly reduced tumor growth. Collectively, this study identifies FLCs as a ligand in the pro-tumorigenic activation of mast cells. Blocking this pathway may open new avenues for the inhibition of tumor growth, while immunohistochemical staining of FLC may be helpful in the diagnosis and prognosis of cancer.

Aim: The conventional clinical formulation of paclitaxel (PTX), Taxol®, consists of Cremophor® EL (CrEL) and ethanol. CrEL-formulated PTX is associated with acute hypersensitivity reactions, anemia and cardiovascular events. In this study, the authors investigated the effects of CrEL-PTX on red blood cells (RBCs) and compared these with the effects observed after exposure to the novel nanoparticle albumin-bound PTX, marketed as Abraxane®., Results: The authors demonstrate that CrEL is primarily responsible for RBC lysis and induction of phosphatidylserine exposure. Phosphatidylserine-exposing RBCs showed increased association with endothelial cells in culture. The authors also identified CrEL as being responsible for vesiculation of RBCs. This is the first time that excipients have been shown to be involved in microvesicle formation. Microvesicles were taken up by endothelial cells., Conclusion: These results offer new insights into the side effect profile of Taxol, which is likely to have implications for patients with erythrocyte disorders. Abraxane did not induce any of these effects on RBCs, indicating that the choice of excipients can have a pronounced influence on the efficacy and side effects of drug molecules.

Background: Phosphatidylserine exposure by red blood cells is acknowledged as a signal that initiates phagocytic removal of the cells from the circulation. Several disorders and conditions are known to induce phosphatidylserine exposure. Removal of phosphatidylserine-exposing red blood cells generally occurs by macrophages in the spleen and liver. Previously, however, we have shown that endothelial cells are also capable of erythrophagocytosis. Key players in the erythrophagocytosis by endothelial cells appeared to be lactadherin and α(v)-integrin. Phagocytosis via the phosphatidylserine-lactadherin-α(v)-integrin pathway is the acknowledged route for removal of apoptotic innate cells by phagocytes., Design and Methods: Endothelial cell phagocytosis of red blood cells was further explored using a more (patho)physiological approach. Red blood cells were exposed to oxidative stress, induced by tert-butyl hydroperoxide. After opsonization with lactadherin, red blood cells were incubated with endothelial cells to study erythrophagocytosis and examine cytotoxicity., Results: Red blood cells exposed to oxidative stress show alterations such as phosphatidylserine exposure and loss of deformability. When incubated with endothelial cells, marked erythrophagocytosis occurred in the presence of lactadherin under both static and flow conditions. As a consequence, intracellular organization was disturbed and endothelial cells were seen to change shape ('rounding up'). Increased expression of apoptotic markers indicated that marked erythrophagocytosis has cytotoxic effects., Conclusions: Activated endothelial cells show significant phagocytosis of phosphatidylserine-exposing and rigid red blood cells under both static and flow conditions. This results in a certain degree of cytotoxicity. We postulate that activated endothelial cells play a role in red blood cell clearance in vivo. Significant erythrophagocytosis can induce endothelial cell loss, which may contribute to vasopathological effects as seen, for instance, in sickle cell disease.

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

The chronic inflammatory environment of tumors is a target for novel antitumor therapeutic strategies. Besides cholesterol lowering effects, statins have been studied for their anti-inflammatory and immunomodulatory properties. These pleiotropic effects result mainly from the altered post-translational modification of GTP-binding proteins which regulate many intracellular pathways involved in cell growth and survival. Although pre-clinical studies suggest that statins may be effective anticancer agents required doses that are 100 to 500 fold higher than those needed to lower cholesterol levels. Furthermore, in view of their wide-ranging effects on cellular metabolism, target site-specific delivery is preferred. In this study, we investigated tumor-specific delivery of pravastatin using small long-circulating liposomes. In vitro studies on the effects of (liposomal) pravastatin on viability and proliferation of tumor cells, endothelial cells and macrophages revealed that the latter were the most sensitive cell type towards (liposomal) pravastatin treatment. In vivo, liposome-encapsulated pravastatin (5mg/kg) inhibited murine B16F10-melanoma growth over 70% as compared to free pravastatin, which was ineffective. As expected, treatments did not influence serum cholesterol levels within the time frame of the study. At 48 h post-injection, 3 μg of pravastatin could still be recovered from the tumors of liposomal pravastatin treated mice, whereas pravastatin could not be detected in tumors of the free drug treated mice (i.e. < 20 ng). In contrast to the free drug, liposomal pravastatin treatment effectively inhibited the production of several pro-inflammatory/pro-angiogenic mediators involved in inflammation and angiogenesis, out of a range of a panel of 24 proteins studied. Furthermore, liposomal pravastatin treatment increased MHC class I protein expression in the tumor tissue whereas free drug showed no effect. Taken together, targeted delivery of statins can improve their tumor growth inhibiting activity by increasing local drug concentration and direct modulation of macrophage function. The antitumor activity seems to result primarily from a local inhibition of tumor inflammation and stimulation of antitumor immune response., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Importance of the Field: Solid tumors rely on efficient oxygen and nutrients transport for their growth, development and survival. Many tumors can stimulate new blood vessel formation. Because this angiogenic vasculature is aberrant from normal host vasculature, several strategies have been explored that specifically target tumor blood vessels., Areas Covered in This Review: Over the past decade, many molecules that act on tumor vasculature have been identified. They can be divided into three groups based on their mechanism of action: i) antiangiogenic molecules cause tumor growth arrest; ii) vasoactive agents induce hyperabnormalization of the tumor vasculature, improving conventional drug accumulation in the tumor; iii) vascular disrupting agents that cause blood vessel congestion, resulting in massive secondary tumor cell necrosis. Many investigational drugs from these classes are currently being evaluated to assess their role in tumor therapy., What the Reader Will Gain: The underlying principle of each of the strategies is discussed, and the (pre)clinical results of the investigational drugs in this class are highlighted., Take Home Message: To fully exploit the therapeutic potential of these drugs, it appears necessary to combine them with conventional anticancer agents, improve their selectivity for tumor vasculature, and develop biomarkers that predict the tumor sensitivity for these vascular strategies.

Objective: Angiogenic endothelial cells can function as phagocytes, and phagocytosis is initiated via the opsonin lactadherin. In this study, we examined the interaction between lactadherin-opsonized erythrocytes with reduced deformability and angiogenic endothelium, as loss of deformability is characteristic for suicidal and aged erythrocytes., Materials and Methods: We used the Arg-Gly-Asp (RGD)-modified erythrocyte model and investigated the deformability parameter by cross-linking erythrocyte membranes through treatment with glutaraldehyde. Association in vitro with primary endothelial cells was detected by flow cytometry and visualized by light, fluorescent, and electron microscopy. Involvement of two crucial factors in phagocytosis, alpha(v)-integrins and Rho guanosine triphosphatase family member Rac1, was studied using small interfering RNA technology. Modified erythrocytes were administered in vivo into tumor-bearing mice to detect phagocytosis by endothelial cells., Results: Glutaraldehyde-treated (rigid) RGD-modified erythrocytes showed a strongly enhanced endothelial cell association compared to flexible RGD-modified erythrocytes. Knockdown by small interfering RNA lipoplexes of alpha(v)-integrins and Rac1 confirmed classical tethering and internalization of rigid RGD-erythrocytes. Upon in vivo administration, tumor endothelium showed pronounced erythrophagocytosis., Conclusion: The pronounced phagocytosis of opsonized erythrocytes with reduced deformability by angiogenic growth factor-activated endothelial cells evokes new insights in endothelial cell function and suggests a role for these endothelial cells in (hematological) disorders because of their capacity to clear disordered erythrocytes.

Vascular disrupting agents (VDAs) are able to affect selectively tumour endothelial cell morphology resulting in vessel occlusion and widespread tumour cell necrosis. However, single-agent antitumour activity of VDAs is typically limited, as tumour regrowth occurs rapidly following drug treatment. To improve the therapeutic effectiveness of VDAs, we investigated liposomal targeting using ZD6126 as a model VDA. ZD6126 is a phosphate-prodrug of the tubulin-binding vascular disrupting agent ZD6126 phenol. ZD6126 was encapsulated into long circulating PEG-liposomes for passive targeting and PEG-liposomes conjugated with peptide ligands containing the RGD-motif for active targeting to alpha(v)-integrins on tumour endothelial cells. ZD6126 could be stably encapsulated, and liposomes displayed minimal leakage in vitro (<10% in 3 weeks). In vivo, upon intravenous injection, free ZD6126 was rapidly converted into ZD6126 phenol, which was cleared from the circulation within minutes. In contrast, ZD6126 encapsulated into either RGD-targeted or PEG liposomes showed prolonged blood circulation times (t(1/2)=10 h), and ZD6126 phenol exposure was also prolonged (t(1/2)=8 h). Both liposomal formulations displayed tumour accumulation plus hepatosplenic uptake by local macrophages. The altered pharmacokinetics and tissue distribution profiles of both liposomal ZD6126 formulations resulted both in single-dose and multiple-dose regimes, in improved therapeutic efficacy in established murine B16.F10 melanomas compared with free ZD6126. The passively and actively targeted liposomes showed equal antitumour efficacy, indicating that delivery of ZD6126 to the tumour tissue may suffice to disrupt tumour blood vessels without the need for specific targeting to the tumour endothelium.

Angiogenic endothelium plays a crucial role in tumor growth. During angiogenesis, complex alterations in the microenvironment occur. In response, the endothelium undergoes phenotypic changes, for example overexpression of alpha(v)-integrins. Here, we show that the overexpression of alpha(v)-integrins on angiogenic endothelial cells is engaged in phagocytic actions involving binding ("tethering") and uptake ("tickling") of lactadherin (also termed MFG-E8)--opsonized particles. Phosphatidylserine (PS)--exposing multilamellar vesicles, "aged" erythrocytes, and apoptotic melanoma cells incubated with lactadherin were all phagocytosed by angiogenic endothelial cells in vitro. Furthermore, we demonstrated lactadherin expression in and around tumor blood vessels making opsonization in situ plausible. By engineering the surface of erythrocytes with covalently coupled cyclic Arg-Gly-Asp (RGD) peptides--mimicking lactadherin opsonization--we could induce phagocytosis by angiogenic endothelial cells both in vitro and in vivo. In vitro, this was confirmed by cytochalasin D preincubation. When RGD-erythrocytes were administered intravenously in tumor-bearing mice, blood vessel congestion followed by tumor core necrosis was seen. Moreover, RGD-erythrocytes could delay tumor growth in a murine melanoma model, possibly through induction of tumor infarctions. These results reveal that angiogenic endothelial cells have phagocytic properties for lactadherin-opsonized large particles and apoptotic cells. Implications of our findings for diagnostic and therapy of angiogenesis-driven diseases are discussed.

Prednisolone disodium phosphate (PLP) encapsulated in long-circulating liposomes (LCL) (LCL-PLP) inhibited tumor growth by 80-90% after a single dose of 20 mg/kg, whereas PLP in the free form was completely ineffective at the same single dose. To generalize our findings with LCL-PLP, the antitumor activity and side effects of LCL containing synthetic glucocorticoids (LCL-GC) other than PLP were investigated. In addition to PLP, budesonide disodium phosphate, dexamethasone disodium phosphate, and methylprednisolone disodium phosphate were selected based on the difference in their potency to activate the human glucocorticoid receptor. The present study shows that the tumor localization of each GC is governed by the transport capacity of the LCL composed of dipalmitoylphosphatidylcholine, cholesterol, and polyethylene glycol 2000-distearoylphosphatidylethanolamine in a molar ratio of 1.85:1.0:0.15. The antitumor potency of the LCL-GC strongly depends on the potency of the type of GC encapsulated. LCL-encapsulated budesonide disodium phosphate (LCL-BUP) had the highest antitumor activity which is likely due to the much higher potency of BUP encapsulated in LCL versus the other three GC types. The high potency of LCL-BUP confers the risk for occurrence of strong side effects. However, at the dose of 3 mg/kg, LCL-BUP was highly efficacious without the occurrence of adverse effects.

Diabetic retinopathy is a secondary complication of hyperglycemia caused by diabetes mellitus. The damage to the retina can ultimately cause vision loss as a result of increased capillary permeability and angiogenesis. Recent progress in the understanding of the mediators that drive angiogenesis, as well as the phenotypes of cells that are involved in this process, has provided a multitude of targets for pharmacologic intervention. This review presents the inhibitors of the biochemical processes that are at the root of diabetic retinopathy (i.e., non-enzymatic glycosylation of biomolecules, oxidative stress, activation of aldose reductase and activation of protein kinase C by formation of diacylglycerol) in addition to the inhibitors of the mechanical damage (i.e., increased vascular permeability, capillary occlusion and neovascularization).

Purpose: Previously, we have shown that complexes of plasmid DNA with the biodegradable polymer poly(2-dimethylamino ethylamino)phosphazene (p(DMAEA)-ppz) mediated tumor selective gene expression after intravenous administration in mice. In this study, we investigated the effect of p(DMAEA)-ppz molecular weight on both in vitro and in vivo tumor transfection, as well as on complex induced toxicity., Materials and Methods: p(DMAEA)-ppz with a broad molar mass distribution was fractionated by preparative size exclusion chromatography. Polyplexes consisting of plasmid DNA and the collected polymer fractions were tested for biophysical properties, (cyto)toxicity and transfection activity., Results: Four p(DMAEA)-ppz fractions were collected with weight average molecular weights ranging from 130 to 950 kDa, and with narrow molecular mass distributions (Mw/Mn from 1.1 to 1.3). At polymer-to-DNA (N/P) ratios above 6, polyplexes based on these polymers were all positively charged (zeta potential 25-29 mV), and had a size of 80-90 nm. The in vitro cytotoxicity of the polyplexes positively correlated with polymer molecular weight. The in vitro transfection activity of the different polyplexes depended on their N/P ratio, and was affected by the degree of cytotoxicity, as well as the colloidal stability of the different polyplexes. Intravenous administration of polyplexes based on the high molecular weight polymers led to apparent toxicity, as a result of polyplex-induced erythrocyte aggregation. On the other hand, administration of polyplexes based on low molecular weight p(DMAEA)-ppz's (Mw 130 kDa) did not show signs of toxicity and resulted in tumor selective gene expression., Conclusion: Polymer molecular weight fractionation enabled us to optimize the transfection efficiency/toxicity ratio of p(DMAEA)-ppz polyplexes for in vitro and in vivo tumor transfection.

Prednisolone phosphate (PLP) encapsulated in long-circulating liposomes can inhibit tumor growth after intravenous administration (i.v.). These antitumor effects of liposomal PLP are the result of the tumor-targeting property of the liposome formulation. The mechanism by which liposomal PLP inhibits tumor growth is unclear. We investigated the effects of liposome-encapsulated PLP versus free PLP on angiogenic protein production in tumor tissue in vivo and on viability and proliferation of tumor and endothelial cells in vitro. In vivo, liposomal PLP had a stronger reducing effect on pro-angiogenic protein levels than free PLP, whereas levels of anti-angiogenic proteins were hardly affected. Cell viability was only slightly affected with either treatment. Liposomal PLP had strong anti-proliferative effects on human umbilical vein endothelial cells, whereas free PLP had hardly any effect. Taken together, the present study points to a strong inhibitory effect of liposomal PLP on tumor angiogenesis by reduction of the intratumoral production of the majority of pro-angiogenic factors studied and direct inhibition of endothelial cell proliferation, which is the result of high prolonged levels of prednisolone in the tumor by liposomal delivery.

Nebulized amphotericin B (AMB) combined with intravenous AMB was studied in persistently leukopenic rats with invasive pulmonary aspergillosis. Pulmonary concentrations of AMB after aerosol treatment were substantially higher than after intravenous liposomal AMB. Nebulized liposomal AMB in addition to intravenous AMB resulted in significantly prolonged survival compared to controls.

Because little is known about the pathophysiology of invasive pulmonary aspergillosis (IPA), we examined changes in pulmonary and general physiology during this disease in an animal model. In a model of fatal left-sided IPA, 19 persistently neutropenic rats were monitored for clinical signs including body temperature, body weight and respiratory distress. A separate group of nine rats with IPA was used for measurements of arterial blood pressure, arterial O2 and CO2 pressure, lung compliance and surfactant function. Body temperature and body weight decreased, whereas respiratory distress increased during progression of the disease. Compared to uninfected controls, in rats with IPA arterial blood pressure and lung compliance were significantly lower, and left lung minimal surface tension was significantly higher. Right lung surfactant function was not affected. Arterial O2 and CO2 pressures were not different between rats with IPA and uninfected controls. Infection with Aspergillus fumigatus in neutropenic rats resulted in hypothermia, body weight loss and respiratory distress. Loss of left lung function was probably compensated by the uninfected right lung, even in a late stage of the disease. Circulatory failure was a major feature in the terminal phase of the infection.

The recently developed siRNA oligonucleotides are an attractive alternative to antisense as a therapeutic modality because of their robust, gene selective silencing of drug target protein expression. To achieve therapeutic success, however, several hurdles must be overcome including rapid clearance, nuclease degradation, and inefficient intracellular localization. In this presentation, we discuss design strategies for development of self-assembling nanoscale carriers for neovasculature targeted delivery of siRNA inhibiting tumor or ocular angiogenesis.

The nebulization of amphotericin B desoxycholate (AMB-DOC), liposomal amphotericin B (L-AMB), amphotericin B lipid complex (ABLC) and amphotericin B colloidal dispersion (ABCD) has been investigated. Particle sizes of generated aerosol droplets were measured. Pulmonary amphotericin B deposition and amphotericin B concentration in blood directly after nebulization and at six weeks after nebulization was measured in healthy rats. The efficacy of nebulized amphotericin B formulations was evaluated in persistently granulocytopenic rats with invasive pulmonary aspergillosis. Treatment was given either after or before fungal inoculation. The endpoint was survival of animals. Aerosol particle sizes, expressed as the values for the mass median diameter were 1.38, 2.43, 0.90 and 2.29 microm for AMB-DOC, L-AMB, ABLC and ABCD, respectively. Amphotericin B concentrations in the lungs directly after nebulization exceeded the minimum inhibitory concentration of Aspergillus fumigatus and amphotericin B was still detected in lungs of rats at six weeks after nebulization. Treatment, started at 16 h after fungal inoculation, resulted in a significantly prolonged survival as compared with sham-treated rats for all four formulations. Prophylactic treatment at one week before fungal inoculation resulted in a significantly prolonged survival for all four formulations. Aerosol treatment given at two weeks before inoculation was effective only for AMB-DOC and L-AMB, whereas treatment given at six weeks resulted in a significantly prolonged survival for L-AMB only. All commercially available amphotericin B preparations could be nebulized efficiently and may be of value in the prophylactic treatment of invasive pulmonary aspergillosis.

Active targeting of angiogenic vasculature represents a promising therapeutic strategy to fight a variety of diseases. In particular, attention has been focused on inhibition of tumor growth. In this review, the recent progress in targeting liposomes to angiogenic endothelial cells is discussed.

Glucocorticoids can inhibit solid tumor growth possibly due to an inhibitory effect on angiogenesis. The antitumor effects of the free drugs have only been observed using treatment schedules based on high and frequent dosing for prolonged periods of time. As long-circulating liposomes accumulate at sites of malignancy, we investigated the tumor-inhibiting potential of liposome-encapsulated prednisolone phosphate. Liposomal prednisolone phosphate could inhibit tumor growth dose-dependently, with 80% to 90% tumor growth inhibition of subcutaneous B16.F10 melanoma and C26 colon carcinoma murine tumor models at 20 mg/kg by single or weekly doses. Prednisolone phosphate in the free form was completely ineffective at this low-frequency treatment schedule, even when administered at a dose of 50 mg/kg. In vitro studies did not show an inhibitory effect of prednisolone (phosphate) on tumor cell, nor on endothelial cell proliferation. Histologic evaluation revealed that liposomal prednisolone phosphate-treated tumors contained a center with areas of picnotic/necrotic cells, which were not apparent in untreated tumors or tumors treated with the free drug. In conclusion, the present study shows potent antitumor effects of liposomal formulations of glucocorticoids in a low dose and low-frequency schedule, offering promise for liposomal glucocorticoids as novel antitumor agents.

Purpose: We purposed to design a cationic polymer that binds to pDNA to form polyplexes and that subsequently degrades within a few days at physiological pH and temperature, releasing the DNA in the cytosol of a cell., Methods: We synthesized a new monomer carbonic acid 2-dimethylamino-ethyl ester 1-methyl-2-(2-methacryloylamino)-ethyl ester (abbreviated HPMA-DMAE) and the corresponding polymer. Hydrolysis of the carbonate ester of both the monomer and the polymer was investigated at 37 degrees C. The DNA condensing properties of the pHPMA-DMAE was studied using dynamic light scattering (DLS) and zeta potential measurements. Degradation of the polyplexes at 37 degrees C and pH 7.4 was monitored with DLS and gel electrophoresis. In vitro transfections were performed in COS-7 cell line., Results: pHPMA-DMAE is able to condense DNA into small particles (110 nm) with a positive zeta potential. The half-life of the polymer and monomer at 37 degrees C and pH 7.4 was around 10 h whereas at pH 5, the half-life was 380 h. In line with this, due to hydrolysis of the side groups, pHPMA-DMAE-based polyplexes dramatically increased in size at 37 degrees C and pH 7.4 whereas at pH 5.0, only a very small increase was observed. Interestingly, intact DNA was released from the polyplexes after 48 h at pH 7.4 whereas all DNA remained bound to the polymer at pH 5.0. Polyplexes were able to transfect cells with minimal cytotoxicity if the endosomal membrane-disrupting peptide INF-7 was added to the polyplex formulation., Conclusions: Degradation of the cationic side-chains of a polymer is a new tool for time-controlled release of DNA from polyplexes, preferably within the cytosol and/or nucleus.

Objective: The kinetics of various parameters of fungal load and cytokines were investigated, in order to acquire insight into the pathogenesis of invasive pulmonary aspergillosis (IPA) during antifungal treatment with amphotericin B., Methods: Neutropenic rats with left-sided IPA received either treatment with amphotericin B or remained untreated. At 0, 4, 8, 16, 24, 48, 72 and 120 h after fungal inoculation, the rats were dissected. The size of the macroscopic pulmonary lesions, the number of cfu and amounts of chitin were determined in the infected left lung. Galactomannan concentrations were measured both in the left lung and serum. The cytokines tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, interferon (IFN)-gamma, IL-4, IL-10, and the chemokines macrophage inflammatory protein (MIP)-2 and monocyte chemoattractant protein (MCP)-1 were determined quantitatively by ELISA in the infected left lung, uninfected right lung and serum., Results: Amphotericin B treatment of IPA resulted in changed aspect of pulmonary lesions and significantly reduced levels of left lung chitin (72 and 120 h), left lung galactomannan (72 and 120 h) and serum galactomannan (120 h), but not left lung cfu, compared with untreated infected rats. In addition, amphotericin B treatment resulted in a significant decrease in levels of left lung IL-6 (at 72 and 120 h), MIP-2 (at 120 h) and MCP-1 (at 120 h). No local or systemic increases in TNF-alpha, IL-1beta or IFN-gamma were observed during infection., Conclusion: It is concluded that treatment with amphotericin B results in decreased fungal load in the infected lung. This reduction in fungal load probably results in a decreased local inflammatory response, as measured by decreased levels of IL-6, MIP-2 and MCP-1 in the infected lung.

Angiogenesis is a key process in the growth and metastasis of a tumor. Disrupting this process is considered a promising treatment strategy. Therefore, a drug delivery system specifically aiming at angiogenic tumor endothelial cells was developed. Alpha v beta 3-integrins are overexpressed on actively proliferating endothelium and represent a possible target. For this, RGD-peptides with affinity for this integrin were coupled to the distal end of poly(ethylene glycol)-coated long-circulating liposomes (LCL) to obtain a stable long-circulating drug delivery system functioning as a platform for multivalent interaction with alpha v beta 3-integrins. The results show that cyclic RGD-peptide-modified LCL exhibited increased binding to endothelial cells in vitro. Moreover, intravital microscopy demonstrated a specific interaction of these liposomes with tumor vasculature, a characteristic not observed for LCL. RGD-LCL encapsulating doxorubicin inhibited tumor growth in a doxorubicin-insensitive murine C26 colon carcinoma model, whereas doxorubicin in LCL failed to decelerate tumor growth. In conclusion, coupling of RGD to LCL redirected these liposomes to angiogenic endothelial cells in vitro and in vivo. RGD-LCL containing doxorubicin showed superior efficacy over non-targeted LCL in inhibiting C26 doxorubicin-insensitive tumor outgrowth. Likely, these RGD-LCL-doxorubicin antitumor effects are brought about through direct effects on tumor endothelial cells.

In common with a proportion of patients with invasive pulmonary aspergillosis (IPA), the efficacy of AmBisome treatment regimens in our rat model remains suboptimal. To investigate whether this might be the result of initially low antifungal activity of amphotericin B at the site of infection when administered in the liposomal form, Fungizone was added to AmBisome at the start of treatment. Groups of granulocytopenic rats with left-sided IPA received 10 day treatment regimens with either AmBisome 10 mg/kg/day (n = 25) or AmBisome 10 mg/kg/day combined with a single dose of Fungizone 1 mg/kg at day 1 (n = 27). Parameters of treatment response included survival, serum galactomannan (GM), size and quality of pulmonary macroscopic lesions, lung weight, viable fungal counts (cfu) and chitin content of the infected lung, and extra-pulmonary disseminated fungal infection. In a separate experiment the significance of early start of treatment to obtain therapeutic efficacy was investigated. Compared with untreated controls, both treatment regimens showed a significant increase in survival and change in parameters of fungal infection except left lung cfu. The combination treatment showed a significant increase in survival compared with AmBisome monotherapy (P = 0.02) and a significant decrease in left lung chitin content (P = 0.03). Differences in circulating GM concentrations between the two treatment regimes approached significance (P = 0.06). Delay in the start of treatment from 16 to 24 h after fungal inoculation resulted in a significant decrease in therapeutic efficacy (P = 0.02). It is concluded that the efficacy of AmBisome therapy can be enhanced by the addition of Fungizone at the start of treatment. This is probably a result of active amphotericin B being immediately available in the lung at the start of treatment.

The value of scintigraphic imaging using 99mTc labeled poly(ethyleneglycol) (PEG) -liposomes for detecting invasive pulmonary aspergillosis at different stages of the disease was investigated in a rat model. At 24, 48, 72, 120 and 168 h after fungal inoculation scintigraphic images were obtained and biodistribution of the radiolabel was determined. Findings were compared with serum galactomannan detection and other parameters of progression of fungal infection. At 48 h liposomal uptake in the infected left lung was increased significantly and 82% of the scintigraphic images was assessed positive. Serum galactomannan was only detected at 72 h and later. Liposomal uptake in the infected left lung increased over time and was significantly correlated with both the size of the pulmonary hemorrhagic lesion and the levels of circulating galactomannan. It was concluded that scintigraphic imaging using 99mTc-PEG-liposomes allows early detection of invasive pulmonary aspergillosis in this model, and that liposomal uptake in the infected lung was strongly associated with the severity of the disease.