Trained Immunity-Promoting Nanobiologic Therapy Suppresses Tumor Growth and Potentiates Checkpoint Inhibition.

Trained immunity, a functional state of myeloid cells, has been proposed as a compelling immune-oncological target. Its efficient induction requires direct engagement of myeloid progenitors in the bone marrow. For this purpose, we developed a bone marrow-avid nanobiologic platform designed specifically to induce trained immunity. We established the potent anti-tumor capabilities of our lead candidate MTP10-HDL in a B16F10 mouse melanoma model. These anti-tumor effects result from trained immunity-induced myelopoiesis caused by epigenetic rewiring of multipotent progenitors in the bone marrow, which overcomes the immunosuppressive tumor microenvironment. Furthermore, MTP10-HDL nanotherapy potentiates checkpoint inhibition in this melanoma model refractory to anti-PD-1 and anti-CTLA-4 therapy. Finally, we determined MTP10-HDL's favorable biodistribution and safety profile in non-human primates. In conclusion, we show that rationally designed nanobiologics can promote trained immunity and elicit a durable anti-tumor response either as a monotherapy or in combination with checkpoint inhibitor drugs.

Inhibiting Inflammation with Myeloid Cell-Specific Nanobiologics Promotes Organ Transplant Acceptance.

Inducing graft acceptance without chronic immunosuppression remains an elusive goal in organ transplantation. Using an experimental transplantation mouse model, we demonstrate that local macrophage activation through dectin-1 and toll-like receptor 4 (TLR4) drives trained immunity-associated cytokine production during allograft rejection. We conducted nanoimmunotherapeutic studies and found that a short-term mTOR-specific high-density lipoprotein (HDL) nanobiologic treatment (mTORi-HDL) averted macrophage aerobic glycolysis and the epigenetic modifications underlying inflammatory cytokine production. The resulting regulatory macrophages prevented alloreactive CD8+ T cell-mediated immunity and promoted tolerogenic CD4+ regulatory T (Treg) cell expansion. To enhance therapeutic efficacy, we complemented the mTORi-HDL treatment with a CD40-TRAF6-specific nanobiologic (TRAF6i-HDL) that inhibits co-stimulation. This synergistic nanoimmunotherapy resulted in indefinite allograft survival. Together, we show that HDL-based nanoimmunotherapy can be employed to control macrophage function in vivo. Our strategy, focused on preventing inflammatory innate immune responses, provides a framework for developing targeted therapies that promote immunological tolerance.

Non-invasive MR assessment of macroscopic and microscopic vascular abnormalities in the rectal tumour-surrounding mesorectum.

OBJECTIVES: To evaluate the MRI macroscopic and microscopic parameters of mesorectal vasculature in rectal cancer patients. METHODS: Thirteen patients with rectal adenocarcinoma underwent a dynamic contrast-enhanced MRI at 1.5 T using a blood pool agent at the primary staging. Mesorectal macrovascular features, i.e., the number of vascular branches, average diameter and length, were assessed from baseline-subtracted post-contrast images by two independent readers. Mesorectal microvascular function was investigated by means of area under the enhancement-time curve (AUC). Histopathology served as reference standard of the tumour response to CRT. RESULTS: The average vessel branching in the mesorectum around the tumour and normal rectal wall was 8.2 ± 3.8 and 1.7 ± 1.3, respectively (reader1: p = 0.001, reader2: p = 0.002). Similarly, the tumour-surrounding mesorectum displayed circa tenfold elevated AUC (p = 0.01). Interestingly, patients with primary node involvement had a twofold higher number of macrovascular branches compared to those with healthy nodes (reader1: p = 0.005 and reader2: p = 0.03). A similar difference was observed between good and poor responders to CRT, whose tumour-surrounding mesorectum displayed 10.7 ± 3.4 and 5.6 ± 1.5 vessels, respectively (reader1/reader2: p = 0.02). CONCLUSIONS: We showed at baseline MRI of rectal tumours a significantly enhanced macrovascular structure and microvascular function in rectal tumour-surrounding mesorectum, and the association of primary mesorectal macrovascular parameters with node involvement and therapy response. KEY POINTS: • Vascular MRI reveals macrovascular and microvascular abnormalities in the rectal tumour-surrounding mesorectum. • Formation of highly vascular stroma precedes the actual tumour invasion. • High macrovascular parameters are associated with node involvement. • Mesorectal vascular network differs for good and poor responders.

Multifunctional magnetic resonance imaging probes.

Magnetic resonance imaging (MRI) is a key imaging modality in cancer diagnostics and therapy monitoring. MRI-based tumor detection and characterization is commonly achieved by exploiting the compositional, metabolic, cellular, and vascular differences between malignant and healthy tissue. Contrast agents are frequently applied to enhance this contrast. The last decade has witnessed an increasing interest in novel multifunctional MRI probes. These multifunctional constructs, often of nanoparticle design, allow the incorporation of multiple imaging agents for complementary imaging modalities as well as anti-cancer drugs for therapeutic purposes. The composition, size, and surface properties of such constructs can be tailored as to improve biodistribution and ensure optimal delivery to the tumor microenvironment by passive or targeted mechanisms. Multifunctional MRI probes hold great promise to facilitate more specific tumor diagnosis, patient-specific treatment planning, the monitoring of local drug delivery, and the early evaluation of therapy. This chapter reviews the state-of-the-art and new developments in the application of multifunctional MRI probes in oncology.

T2 weighted signal intensity evolution may predict pathological complete response after treatment for rectal cancer.

OBJECTIVES: To determine the diagnostic value of T2-weighted signal intensity evolution in the tumour for detection of complete response to neoadjuvant chemoradiotherapy in patients with rectal cancer. METHODS: Thirty-nine patients diagnosed with locally advanced adenocarcinoma and treated with chemoradiotherapy (CRT), followed by surgery, underwent magnetic resonance imaging (MRI) before and after CRT on 1.5-T MRI using T2-weighted fast spin-echo (FSE) imaging. The relative T2-weighted signal intensity (rT2wSI) distribution in the tumour and post-CRT residual tissue was characterised by means of the descriptive statistical parameters, such as the mean, 95th percentile and standard deviation (SD). Receiver operating characteristic curves were used to determine the diagnostic potential of the CRT-induced alterations (Δ) in rT2wSI descriptives. The tumour regression grade (TRG) served as a histopathological reference standard. RESULTS: CRT induced a significant decrease of approximately 50% in all rT2wSI descriptives in complete responders (TRG1). This drop was significantly larger than for incomplete response groups (TRG2-TRG4). The ΔrT2wSI descriptives produced a high diagnostic performance for identification of complete responders, e.g. Δ95th percentile, ΔSD and Δmean resulted in accuracy of 92%, 90% and 82%, respectively. CONCLUSIONS: Quantitative assessment of the CRT-induced changes in the tumour T2-weighted signal intensity provides high diagnostic performance for selection of complete responders.

Spatial Transcriptional Mapping Reveals Site-Specific Pathways Underlying Human Atherosclerotic Plaque Rupture.

BACKGROUND: Atherosclerotic plaque ruptures, triggered by blood flow-associated biomechanical forces, cause most myocardial infarctions and strokes. OBJECTIVES: This study aims to investigate the exact location and underlying mechanisms of atherosclerotic plaque ruptures, identifying therapeutic targets against cardiovascular events. METHODS: Histology, electron microscopy, bulk and spatial RNA sequencing on human carotid plaques were studied in proximal, most stenotic, and distal regions along the longitudinal blood flow direction. Genome-wide association studies were used to examine heritability enrichment and causal relationships of atherosclerosis and stroke. Associations between top differentially expressed genes (DEGs) and preoperative and postoperative cardiovascular events were examined in a validation cohort. RESULTS: In human carotid atherosclerotic plaques, ruptures predominantly occurred in the proximal and most stenotic regions but not in the distal region. Histologic and electron microscopic examination showed that proximal and most stenotic regions exhibited features of plaque vulnerability and thrombosis. RNA sequencing identified DEGs distinguishing the proximal and most stenotic regions from the distal region which were deemed as most relevant to atherosclerosis-associated diseases as shown by heritability enrichment analyses. The identified pathways associated with the proximal rupture-prone regions were validated by spatial transcriptomics, firstly in human atherosclerosis. Of the 3 top DEGs, matrix metallopeptidase 9 emerged particularly because Mendelian randomization suggested that its high circulating levels were causally associated with atherosclerosis risk. CONCLUSIONS: Our findings show plaque site-specific transcriptional signatures associated with proximal rupture-prone regions of carotid atherosclerotic plaques. This led to the geographical mapping of novel therapeutic targets, such as matrix metallopeptidase 9, against plaque rupture.

Resolving sepsis-induced immunoparalysis via trained immunity by targeting interleukin-4 to myeloid cells.

Immunoparalysis is a compensatory and persistent anti-inflammatory response to trauma, sepsis or another serious insult, which increases the risk of opportunistic infections, morbidity and mortality. Here, we show that in cultured primary human monocytes, interleukin-4 (IL4) inhibits acute inflammation, while simultaneously inducing a long-lasting innate immune memory named trained immunity. To take advantage of this paradoxical IL4 feature in vivo, we developed a fusion protein of apolipoprotein A1 (apoA1) and IL4, which integrates into a lipid nanoparticle. In mice and non-human primates, an intravenously injected apoA1-IL4-embedding nanoparticle targets myeloid-cell-rich haematopoietic organs, in particular, the spleen and bone marrow. We subsequently demonstrate that IL4 nanotherapy resolved immunoparalysis in mice with lipopolysaccharide-induced hyperinflammation, as well as in ex vivo human sepsis models and in experimental endotoxemia. Our findings support the translational development of nanoparticle formulations of apoA1-IL4 for the treatment of patients with sepsis at risk of immunoparalysis-induced complications.

Multi-parametric assessment of the anti-angiogenic effects of liposomal glucocorticoids.

Inflammation plays a prominent role in tumor growth. Anti-inflammatory drugs have therefore been proposed as anti-cancer therapeutics. In this study, we determined the anti-angiogenic activity of a single dose of liposomal prednisolone phosphate (PLP-L), by monitoring tumor vascular function and viability over a period of one week. C57BL/6 mice were inoculated subcutaneously with B16F10 melanoma cells. Six animals were PLP-L-treated and six served as control. Tumor tissue and vascular function were probed using MRI before and at three timepoints after treatment. DCE-MRI was used to determine K(trans), v(e), time-to-peak, initial slope and the fraction of non-enhancing pixels, complemented with immunohistochemistry. The apparent diffusion coefficient (ADC), T(2) and tumor size were assessed with MRI as well. PLP-L treatment resulted in smaller tumors and caused a significant drop in K(trans) 48 h post-treatment, which was maintained until one week after drug administration. However, this effect was not sufficient to significantly distinguish treated from non-treated animals. The therapy did not affect tumor tissue viability but did prevent the ADC decrease observed in the control group. No evidence for PLP-L-induced tumor vessel normalization was found on histology. Treatment with PLP-L altered tumor vascular function. This effect did not fully explain the tumor growth inhibition, suggesting a broader spectrum of PLP-L activities.

Synergistic targeting of alphavbeta3 integrin and galectin-1 with heteromultivalent paramagnetic liposomes for combined MR imaging and treatment of angiogenesis.

Effective and specific targeting of nanoparticles is of paramount importance in the fields of targeted therapeutics and diagnostics. In the current study, we investigated the targeting efficacy of nanoparticles that were functionalized with two angiogenesis-specific targeting ligands, an alpha(v)beta(3) integrin-specific and a galectin-1-specific peptide. We show in vitro, using optical techniques and MRI, that the dual-targeting approach produces synergistic targeting effects, causing a dramatically elevated uptake of nanoparticles as compared to single ligand targeting.

Diverse ultrastructural landscape of atherosclerotic endothelium.

BACKGROUND AND AIMS: The endothelium plays a major role in atherosclerosis, yet the endothelial plaque surface is a largely uncharted territory. Here we hypothesize that atherosclerosis-driven remodeling of the endothelium is a dynamic process, involving both damaging and regenerative mechanisms. METHODS: Using scanning electron microscopy (SEM) and immuno-SEM, we studied endothelial junction ultrastructure, endothelial openings and immune cell-endothelium interactions in eight apoe-/- mice and two human carotid plaques. RESULTS: The surface of early mouse plaques (n = 11) displayed a broad range of morphological alterations, including junctional disruptions and large transcellular endothelial pores with the average diameter between 0.6 and 3 µm. The shoulder region of advanced atherosclerotic lesions (n = 7) had a more aggravated morphology with 8 µm-size paracellular openings at two-fold higher density. In contrast, the central apical surface of advanced plaques, i.e., the plaque body (n = 7), displayed endothelial normalization, as shown by a significantly higher frequency of intact endothelial junctions and a lower incidence of paracellular pores. This normalized endothelial phenotype correlated with low immune cell density (only 5 cells/mm2). The human carotid plaque surface (n = 2) displayed both well-organized and disrupted endothelium with similar features as described above. In addition, they were accompanied by extensive thrombotic areas. CONCLUSIONS: Our study unveils the spectrum of endothelial abnormalities associated with the development of atherosclerosis. These were highly abundant in early lesions and in the shoulder region of advanced plaques, while normalized at the advanced plaque's body. Similar endothelial features were observed in human atherosclerotic plaques, underlining the versatility of endothelial transformations in atherosclerosis.

A modular approach toward producing nanotherapeutics targeting the innate immune system.

Immunotherapies controlling the adaptive immune system are firmly established, but regulating the innate immune system remains much less explored. The intrinsic interactions between nanoparticles and phagocytic myeloid cells make these materials especially suited for engaging the innate immune system. However, developing nanotherapeutics is an elaborate process. Here, we demonstrate a modular approach that facilitates efficiently incorporating a broad variety of drugs in a nanobiologic platform. Using a microfluidic formulation strategy, we produced apolipoprotein A1-based nanobiologics with favorable innate immune system-engaging properties as evaluated by in vivo screening. Subsequently, rapamycin and three small-molecule inhibitors were derivatized with lipophilic promoieties, ensuring their seamless incorporation and efficient retention in nanobiologics. A short regimen of intravenously administered rapamycin-loaded nanobiologics (mTORi-NBs) significantly prolonged allograft survival in a heart transplantation mouse model. Last, we studied mTORi-NB biodistribution in nonhuman primates by PET/MR imaging and evaluated its safety, paving the way for clinical translation.

Prosaposin mediates inflammation in atherosclerosis.

Macrophages play a central role in the pathogenesis of atherosclerosis. The inflammatory properties of these cells are dictated by their metabolism, of which the mechanistic target of rapamycin (mTOR) signaling pathway is a key regulator. Using myeloid cell-specific nanobiologics in apolipoprotein E-deficient (Apoe -/-) mice, we found that targeting the mTOR and ribosomal protein S6 kinase-1 (S6K1) signaling pathways rapidly diminished plaque macrophages' inflammatory activity. By investigating transcriptome modifications, we identified Psap, a gene encoding the lysosomal protein prosaposin, as closely related with mTOR signaling. Subsequent in vitro experiments revealed that Psap inhibition suppressed both glycolysis and oxidative phosphorylation. Transplantation of Psap -/- bone marrow to low-density lipoprotein receptor knockout (Ldlr -/-) mice led to a reduction in atherosclerosis development and plaque inflammation. Last, we confirmed the relationship between PSAP expression and inflammation in human carotid atherosclerotic plaques. Our findings provide mechanistic insights into the development of atherosclerosis and identify prosaposin as a potential therapeutic target.

Paramagnetic and fluorescent liposomes for target-specific imaging and therapy of tumor angiogenesis.

Angiogenesis is essential for tumor growth and metastatic potential and for that reason considered an important target for tumor treatment. Noninvasive imaging technologies, capable of visualizing tumor angiogenesis and evaluating the efficacy of angiostatic therapies, are therefore becoming increasingly important. Among the various imaging modalities, magnetic resonance imaging (MRI) is characterized by a superb spatial resolution and anatomical soft-tissue contrast. Revolutionary advances in contrast agent chemistry have delivered versatile angiogenesis-specific molecular MRI contrast agents. In this paper, we review recent advances in the preclinical application of paramagnetic and fluorescent liposomes for noninvasive visualization of the molecular processes involved in tumor angiogenesis. This liposomal contrast agent platform can be prepared with a high payload of contrast generating material, thereby facilitating its detection, and is equipped with one or more types of targeting ligands for binding to specific molecules expressed at the angiogenic site. Multimodal liposomes endowed with contrast material for complementary imaging technologies, e.g., MRI and optical, can be exploited to gain important preclinical insights into the mechanisms of binding and accumulation at angiogenic vascular endothelium and to corroborate the in vivo findings. Interestingly, liposomes can be designed to contain angiostatic therapeutics, allowing for image-supervised drug delivery and subsequent monitoring of therapeutic efficacy.

Nuclear imaging approaches facilitating nanomedicine translation.

Nanomedicine approaches can effectively modulate the biodistribution and bioavailability of therapeutic agents, improving their therapeutic index. However, despite the ever-increasing amount of literature reporting on preclinical nanomedicine, the number of nanotherapeutics receiving FDA approval remains relatively low. Several barriers exist that hamper the effective preclinical evaluation and clinical translation of nanotherapeutics. Key barriers include insufficient understanding of nanomedicines' in vivo behavior, inadequate translation from murine models to larger animals, and a lack of patient stratification strategies. Integrating quantitative non-invasive imaging techniques in nanomedicine development offers attractive possibilities to address these issues. Among the available imaging techniques, nuclear imaging by positron emission tomography (PET) and single-photon emission computed tomography (SPECT) are highly attractive in this context owing to their quantitative nature and uncontested sensitivity. In basic and translational research, nuclear imaging techniques can provide critical quantitative information about pharmacokinetic parameters, biodistribution profiles or target site accumulation of nanocarriers and their associated payload. During clinical evaluation, nuclear imaging can be used to select patients amenable to nanomedicine treatment. Here, we review how nuclear imaging-based approaches are increasingly being integrated into nanomedicine development and discuss future developments that will accelerate their clinical translation.

Nanoparticle-Aided Characterization of Arterial Endothelial Architecture during Atherosclerosis Progression and Metabolic Therapy.

Atherosclerosis is associated with a compromised endothelial barrier, facilitating the accumulation of immune cells and macromolecules in atherosclerotic lesions. In this study, we investigate endothelial barrier integrity and the enhanced permeability and retention (EPR) effect during atherosclerosis progression and therapy in Apoe-/- mice using hyaluronan nanoparticles (HA-NPs). Utilizing ultrastructural and en face plaque imaging, we uncover a significantly decreased junction continuity in the atherosclerotic plaque-covering endothelium compared to the normal vessel wall, indicative of disrupted endothelial barrier. Intriguingly, the plaque advancement had a positive effect on junction stabilization, which correlated with a 3-fold lower accumulation of in vivo administrated HA-NPs in advanced plaques compared to early counterparts. Furthermore, by using super-resolution and correlative light and electron microscopy, we trace nanoparticles in the plaque microenvironment. We find nanoparticle-enriched endothelial junctions, containing 75% of detected HA-NPs, and a high HA-NP accumulation in the endothelium-underlying extracellular matrix, which suggest an endothelial junctional traffic of HA-NPs to the plague. Finally, we probe the EPR effect by HA-NPs in the context of metabolic therapy with a glycolysis inhibitor, 3PO, proposed as a vascular normalizing strategy. The observed trend of attenuated HA-NP uptake in aortas of 3PO-treated mice coincides with the endothelial silencing activity of 3PO, demonstrated in vitro. Interestingly, the therapy also reduced the plaque inflammatory burden, while activating macrophage metabolism. Our findings shed light on natural limitations of nanoparticle accumulation in atherosclerotic plaques and provide mechanistic insight into nanoparticle trafficking across the atherosclerotic endothelium. Furthermore, our data contribute to the rising field of endothelial barrier modulation in atherosclerosis.

Hyaluronan Nanoparticles Selectively Target Plaque-Associated Macrophages and Improve Plaque Stability in Atherosclerosis.

Hyaluronan is a biologically active polymer, which can be formulated into nanoparticles. In our study, we aimed to probe atherosclerosis-associated inflammation by using hyaluronan nanoparticles and to determine whether they can ameliorate atherosclerosis. Hyaluronan nanoparticles (HA-NPs) were prepared by reacting amine-functionalized oligomeric hyaluronan (HA) with cholanic ester and labeled with a fluorescent or radioactive label. HA-NPs were characterized in vitro by several advanced microscopy methods. The targeting properties and biodistribution of HA-NPs were studied in apoe-/- mice, which received either fluorescent or radiolabeled HA-NPs and were examined ex vivo by flow cytometry or nuclear techniques. Furthermore, three atherosclerotic rabbits received 89Zr-HA-NPs and were imaged by PET/MRI. The therapeutic effects of HA-NPs were studied in apoe-/- mice, which received weekly doses of 50 mg/kg HA-NPs during a 12-week high-fat diet feeding period. Hydrated HA-NPs were ca. 90 nm in diameter and displayed very stable morphology under hydrolysis conditions. Flow cytometry revealed a 6- to 40-fold higher uptake of Cy7-HA-NPs by aortic macrophages compared to normal tissue macrophages. Interestingly, both local and systemic HA-NP-immune cell interactions significantly decreased over the disease progression. 89Zr-HA-NPs-induced radioactivity in atherosclerotic aortas was 30% higher than in wild-type controls. PET imaging of rabbits revealed 6-fold higher standardized uptake values compared to the muscle. The plaques of HA-NP-treated mice contained 30% fewer macrophages compared to control and free HA-treated group. In conclusion, we show favorable targeting properties of HA-NPs, which can be exploited for PET imaging of atherosclerosis-associated inflammation. Furthermore, we demonstrate the anti-inflammatory effects of HA-NPs in atherosclerosis.

Can perfusion MRI predict response to preoperative treatment in rectal cancer?

BACKGROUND AND PURPOSE: Dynamic contrast-enhanced MRI (DCE-MRI) provides information on perfusion and could identify good prognostic tumors. Aim of this study was to evaluate whether DCE-MRI using a novel blood pool contrast-agent can accurately predict the response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer. MATERIALS AND METHODS: Thirty patients underwent DCE-MRI before and 7-10weeks after chemoradiotherapy. Regions of interest were drawn on DCE-MRI with T2W-images as reference. DCE-MRI-based kinetic parameters (initial slope, initial peak, late slope, and AUC at 60, 90, and 120s) determined pre- and post-CRT and their Δ were compared between good (TRG1-2) and poor (TRG3-5) responders. Optimal thresholds were determined and sensitivities, specificities, positive predictive values (PPV), and negative predictive values (NPV) were calculated. RESULTS: Pre-therapy, the late slope was able to discriminate between good and poor responders (-0.05×10(-3) vs. 0.62×10(-3), p<0.001) with an AUC of 0.90, sensitivity 92%, specificity 82%, PPV 80%, and NPV 93%. Other pre-CRT parameters showed no significant differences, nor any post-CRT parameters or their Δ. CONCLUSIONS: The kinetic parameter 'late slope' derived from DCE-MRI could potentially be helpful to predict before the onset of neoadjuvant chemoradiotherapy which tumors are likely going to respond. This could allow for personalized treatment-options in rectal cancer patients.

Dual-targeting of αvß3 and galectin-1 improves the specificity of paramagnetic/fluorescent liposomes to tumor endothelium in vivo.

Molecular imaging of angiogenesis requires a highly specific and efficient contrast agent for targeting activated endothelium. We have previously demonstrated that paramagnetic and fluorescent liposomes functionalized with two angiogenesis-specific ligands, the galectin-1-specific anginex (Anx) and the α(v)ß(3) integrin-specific RGD, produce synergistic targeting effect in vitro. In the current study, we applied Anx and RGD dual-conjugated liposomes (Anx/RGD-L) for angiogenesis-specific MRI in vivo, focusing on the specificity and efficacy of liposome association with tumor endothelium. The targeting properties, clearance kinetics and biodistribution of Anx/RGD-L were investigated in B16F10 melanoma-bearing mice, and compared to liposomes functionalized with either Anx (Anx-L) or RGD (RGD-L). The contrast enhancement produced by dual- and single-targeted nanoparticles in the tumor was measured using in vivo T(1)-weighted MRI, complemented by ex vivo immunohistochemical evaluation of tumor tissues. Blood clearance kinetics of Anx/RGD-L was three-fold more rapid than for RGD-L, but comparable to Anx-L. Both dual- and single-targeted liposomes produced similar changes in MRI contrast parameters in tumors with high inter-tumor variability (ΔR(1)=0.04±0.03s(-1), 24h post-contrast). Importantly, however, the specificity of Anx/RGD-L association with tumor endothelium of 53±6%, assessed by fluorescence microscopy, was significantly higher compared to 43±9% (P=0.043) and 28±8% (P=0.0001) of Anx-L and RGD-L, respectively. In contrast, long-circulating RGD-L were on average 16% more efficient in targeting tumor endothelium compared to Anx/RGD-L. Significant differences were also found in the biodistribution of investigated contrast agents. In conclusion, synergistic targeting of α(v)ß(3) and galectin-1 improved the specificity of the association of the liposomal contrast agent to tumor endothelium in vivo, providing therefore a more reliable MRI readout of the angiogenic activity.

Anti-tumor activity of liposomal glucocorticoids: The relevance of liposome-mediated drug delivery, intratumoral localization and systemic activity.

Tumor-associated inflammation has been recognized as an important tumor growth propagator and, therefore, represents an attractive target for anti-cancer therapy. In the current study, inspired by recent findings on the anti-tumor activity of liposomal glucocorticoids, we introduce paramagnetic and fluorescent liposomes, encapsulating prednisolone phosphate (PLP), to evaluate the local delivery of liposomal glucocorticoids to the tumor and its importance for the therapeutic response. The new multifunctional liposomes (Gd-PLP-L) (120nm diameter, 5.8mg PLP/60µmol lipid, bioexponential blood-clearance kinetics (T(1/2α)=2.4±0.5h, T(1/2ß)=42.0±12.4h), drug leakage of 15%/72h (in vitro)), containing 25mol% Gd-DTPA-lipid and 0.1mol% of rhodamine-lipid, were tested in B16F10 melanoma subcutaneously inoculated in C57BL/6 mice, and compared to the original PLP formulation (PLP-L). A single dose of Gd-PLP-L (20mgPLP/kg/week, i.v.) was found to significantly inhibit tumor growth compared to non-treated mice (P<0.05), similarly to PLP-L. The accumulation efficacy of the liposomal agent in the tumor was assessed with MRI, using the increase in the longitudinal relaxation rate (ΔR(1)) as a marker. Interestingly, large inter-tumor differences in ΔR(1) (0.009-0.063s(-1), 24h post-administration), corresponding to highly variable intratumoral Gd-PLP-L levels, did not correlate to the effectiveness of tumor growth inhibition. Uptake of liposomes by tumor-associated macrophages (TAM), determined by ex-vivo fluorescence microscopy, was limited to only 5% of the TAM population. Furthermore, the therapy did not lead to TAM depletion. Importantly, a 90% drop in white blood cell count both after Gd-PLP-L and PLP-L administration was observed. This depletion may reduce tumor infiltration of monocytes, which stimulate angiogenesis, and, thus, possibly co-contributes to the anti-tumor effects. In conclusion, MRI provides a powerful instrument to monitor the delivery of liposomal therapeutics to tumors and guided us to reveal that the activity of liposomal glucocorticoids is not limited to the tumor site only.