Modulated Electro-Hyperthermia Accelerates Tumor Delivery and Improves Anticancer Activity of Doxorubicin Encapsulated in Lyso-Thermosensitive Liposomes in 4T1-Tumor-Bearing Mice.

Modulated electro-hyperthermia (mEHT) is an adjuvant cancer therapy that enables tumor-selective heating (+2.5 °C). In this study, we investigated whether mEHT accelerates the tumor-specific delivery of doxorubicin (DOX) from lyso-thermosensitive liposomal doxorubicin (LTLD) and improves its anticancer efficacy in mice bearing a triple-negative breast cancer cell line (4T1). The 4T1 cells were orthotopically injected into Balb/C mice, and mEHT was performed on days 9, 12, and 15 after the implantation. DOX, LTLD, or PEGylated liposomal DOX (PLD) were administered for comparison. The tumor size and DOX accumulation in the tumor were measured. The cleaved caspase-3 (cC3) and cell proliferation were evaluated by cC3 or Ki67 immunohistochemistry and Western blot. The LTLD+mEHT combination was more effective at inhibiting tumor growth than the free DOX and PLD, demonstrated by reductions in both the tumor volume and tumor weight. LTLD+mEHT resulted in the highest DOX accumulation in the tumor one hour after treatment. Tumor cell damage was associated with cC3 in the damaged area, and with a reduction in Ki67 in the living area. These changes were significantly the strongest in the LTLD+mEHT-treated tumors. The body weight loss was similar in all mice treated with any DOX formulation, suggesting no difference in toxicity. In conclusion, LTLD combined with mEHT represents a novel approach for DOX delivery into cancer tissue.

Selective tumor antigen vaccine delivery to human CD169+ antigen-presenting cells using ganglioside-liposomes.

Priming of CD8+ T cells by dendritic cells (DCs) is crucial for the generation of effective antitumor immune responses. Here, we describe a liposomal vaccine carrier that delivers tumor antigens to human CD169/Siglec-1+ antigen-presenting cells using gangliosides as targeting ligands. Ganglioside-liposomes specifically bound to CD169 and were internalized by in vitro-generated monocyte-derived DCs (moDCs) and macrophages and by ex vivo-isolated splenic macrophages in a CD169-dependent manner. In blood, high-dimensional reduction analysis revealed that ganglioside-liposomes specifically targeted CD14+ CD169+ monocytes and Axl+ CD169+ DCs. Liposomal codelivery of tumor antigen and Toll-like receptor ligand to CD169+ moDCs and Axl+ CD169+ DCs led to cytokine production and robust cross-presentation and activation of tumor antigen-specific CD8+ T cells. Finally, Axl+ CD169+ DCs were present in cancer patients and efficiently captured ganglioside-liposomes. Our findings demonstrate a nanovaccine platform targeting CD169+ DCs to drive antitumor T cell responses.

Utility of Intravenous Curcumin Nanodelivery Systems for Improving In Vivo Pharmacokinetics and Anticancer Pharmacodynamics.

Curcumin nanoformulations for intravenous injection have been developed to offset poor absorption, biotransformation, degradation, and excessive clearance associated with parenteral delivery. This review investigates (1) whether intravenous nanoformulations improve curcumin pharmacokinetics (PK) and (2) whether improved PK yields greater therapeutic efficacy. Standard PK parameters (measured maximum concentration [Cmax], area under the curve [AUC], distribution volume [Vd], and clearance [CL]) of intravenously administered free curcumin in mice and rats were sourced from literature and compared to curcumin formulated in nanoparticles, micelles, and liposomes. The studies that also featured analysis of pharmacodynamics (PD) in murine cancer models were used to determine whether improved PK of nanoencapsulated curcumin resulted in improved PD. The distribution and clearance of free and nanoformulated curcumin were very fast, typically accounting for >80% curcumin elimination from plasma within 60 min. Case-matched analysis demonstrated that curcumin nanoencapsulation generally improved curcumin PK in terms of measured Cmax (n = 27) and AUC (n = 33), and to a lesser extent Vd and CL. However, when the data were unpaired and clustered for comparative analysis, only 5 out of the 12 analyzed nanoformulations maintained a higher relative curcumin concentration in plasma over time compared to free curcumin. Quantitative analysis of the mean plasma concentration of free curcumin versus nanoformulated curcumin did not reveal an overall marked improvement in curcumin PK. No correlation was found between PK and PD, suggesting that augmentation of the systemic presence of curcumin does not necessarily lead to greater therapeutic efficacy.

Multimodal Positron Emission Tomography Imaging to Quantify Uptake of 89Zr-Labeled Liposomes in the Atherosclerotic Vessel Wall.

Nanotherapy has recently emerged as an experimental treatment option for atherosclerosis. To fulfill its promise, robust noninvasive imaging approaches for subject selection and treatment evaluation are warranted. To that end, we present here a positron emission tomography (PET)-based method for quantification of liposomal nanoparticle uptake in the atherosclerotic vessel wall. We evaluated a modular procedure to label liposomal nanoparticles with the radioisotope zirconium-89 (89Zr). Their biodistribution and vessel wall targeting in a rabbit atherosclerosis model was evaluated up to 15 days after intravenous injection by PET/computed tomography (CT) and PET/magnetic resonance imaging (PET/MRI). Vascular permeability was assessed in vivo using three-dimensional dynamic contrast-enhanced MRI (3D DCE-MRI) and ex vivo using near-infrared fluorescence (NIRF) imaging. The 89Zr-radiolabeled liposomes displayed a biodistribution pattern typical of long-circulating nanoparticles. Importantly, they markedly accumulated in atherosclerotic lesions in the abdominal aorta, as evident on PET/MRI and confirmed by autoradiography, and this uptake moderately correlated with vascular permeability. The method presented herein facilitates the development of nanotherapy for atherosclerotic disease as it provides a tool to screen for nanoparticle targeting in individual subjects' plaques.

A Novel Platform for Cancer Vaccines: Antigen-Selective Delivery to Splenic Marginal Zone B Cells via Repeated Injections of PEGylated Liposomes.

Treating cancer with vaccines has been a challenge. In this study, we introduce a novel Ag delivery platform for cancer vaccines that delivers an encapsulated Ag to splenic marginal zone B (MZ-B) cells via the aid of a PEGylated liposome (PL) system. Splenic MZ-B cells have recently attracted interest as alternative APCs. In mice, preimmunization with empty (no Ag encapsulation) PLs triggered the efficient delivery of a subsequent dose of Ag-containing PLs, injected 3 d later, to the spleen compared with a single dose of Ag-containing PLs. In addition, immunization with empty PLs allowed three subsequent sequential injections of OVA-PLs to efficiently induce a CTL response against OVA-expressing murine thymoma (EG7-OVA) cells and resulted in in vivo growth inhibition of subsequently inoculated EG7-OVA cells. However, these sequential treatments require repeated immunizations to achieve their antitumor effect. Therefore, to improve the antitumor effect of our novel vaccine system, an adjuvant, α-galactosylceramide (αGC), was incorporated into the OVA-PLs (αGC/OVA-PLs). As expected, the incorporation of αGC reduced the required number of immunizations with OVA-PLs to the point that a single immunization treatment with empty PLs and an injection of αGC/OVA-PL efficiently triggered a potent CTL induction, resulting in a rejection of the development and a suppression of the growth of tumors that had already developed s.c. Results of this study indicate that a novel Ag delivery platform that grants efficient Ag delivery to splenic MZ-B cells shows promise as a therapeutic modality for conquering tumor growth and/or progression.

Hyperthermia-triggered release of hypoxic cell radiosensitizers from temperature-sensitive liposomes improves radiotherapy efficacy in vitro.

Hypoxia is a characteristic feature of solid tumors and an important cause of resistance to radiotherapy. Hypoxic cell radiosensitizers have been shown to increase radiotherapy efficacy, but dose-limiting side effects prevent their widespread use in the clinic. We propose the encapsulation of hypoxic cell radiosensitizers in temperature-sensitive liposomes (TSL) to target the radiosensitizers specifically to tumors and to avoid unwanted accumulation in healthy tissues. The main objective of the present study is to develop and characterize TSL loaded with the radiosensitizer pimonidazole (PMZ) and to evaluate the in vitro efficacy of free PMZ and PMZ encapsulated in TSL in combination with hyperthermia and radiotherapy. PMZ was actively loaded into TSL at different drug/lipid ratios, and the physicochemical characteristics and the stability of the resulting TSL-PMZ were evaluated. PMZ release was determined at 37 °C and 42 °C in HEPES buffer saline and fetal bovine serum. The concentration-dependent radiosensitizing effect of PMZ was investigated by exposing FaDu cells to different PMZ concentrations under hypoxic conditions followed by exposure to ionizing irradiation. The efficacy of TSL-PMZ in combination with hyperthermia and radiotherapy was determined in vitro, assessing cell survival and DNA damage by means of the clonogenic assay and histone H2AX phosphorylation, respectively. All TSL-PMZ formulations showed high encapsulation efficiencies and were stable for 30 d upon storage at 4 °C and 20 °C. Fast PMZ release was observed at 42 °C, regardless of the drug/lipid ratio. Increasing the PMZ concentration significantly enhanced the effect of ionizing irradiation. Pre-heated TSL-PMZ in combination with radiotherapy caused a 14.3-fold increase in cell death as compared to radiotherapy treatment alone. In conclusion, our results indicate that TSL-PMZ in combination with hyperthermia can assist in improving the efficacy of radiotherapy under hypoxic conditions.

Liposomal delivery of dexamethasone attenuates prostate cancer bone metastatic tumor growth in vivo.

BACKGROUND: The inflammatory tumor microenvironment, and more specifically the tumor-associated macrophages, plays an essential role in the development and progression of prostate cancer towards metastatic bone disease. Tumors are often characterized by a leaky vasculature, which - combined with the prolonged circulation kinetics of liposomes - leads to efficient tumor localization of these drug carriers, via the so-called enhanced permeability and retention (EPR) -effect. In this study, we evaluated the utility of targeted, liposomal drug delivery of the glucocorticoid dexamethasone in a model of prostate cancer bone metastases. METHODS: Tumor-bearing Balb-c nu/nu mice were treated intravenously with 0.2-1.0-5.0 mg/kg/week free- and liposomal DEX for 3-4 weeks and tumor growth was monitored by bioluminescent imaging. RESULTS: Intravenously administered liposomes localize efficiently to bone metastases in vivo and treatment of established bone metastases with (liposomal) dexamethasone resulted in a significant inhibition of tumor growth up to 26 days after initiation of treatment. Furthermore, 1.0 mg/kg liposomal dexamethasone significantly outperformed 1.0 mg/kg free dexamethasone, and was found to be well-tolerated at clinically-relevant dosages that display potent anti-tumor efficacy. CONCLUSIONS: Liposomal delivery of the glucocorticoid dexamethasone inhibits the growth of malignant bone lesions. We believe that liposomal encapsulation of dexamethasone offers a promising new treatment option for advanced, metastatic prostate cancer which supports further clinical evaluation.

Complement activation as a bioequivalence issue relevant to the development of generic liposomes and other nanoparticulate drugs.

Liposomes are known to activate the complement (C) system, which can lead in vivo to a hypersensitivity syndrome called C activation-related pseudoallergy (CARPA). CARPA has been getting increasing attention as a safety risk of i.v. therapy with liposomes, whose testing is now recommended in bioequivalence evaluations of generic liposomal drug candidates. This review highlights the adverse consequences of C activation, the unique symptoms of CARPA triggered by essentially all i.v. administered liposomal drugs, and the various features of vesicles influencing this adverse immune effect. For the case of Doxil, we also address the mechanism of C activation and the opsonization vs. long circulation (stealth) paradox. In reviewing the methods of assessing C activation and CARPA, we delineate the most sensitive porcine model and an algorithm for stepwise evaluation of the CARPA risk of i.v. liposomes, which are proposed for standardization for preclinical toxicology evaluation of liposomal and other nanoparticulate drug candidates.

Superoxide dismutase enzymosomes: carrier capacity optimization, in vivo behaviour and therapeutic activity.

PURPOSE: A strategy not usually used to improve carrier-mediated delivery of therapeutic enzymes is the attachment of the enzymes to the outer surface of liposomes. The aim of our work was to design a new type of enzymosomes with a sufficient surface-exposed enzyme load while preserving the structural integrity of the liposomal particles and activity of the enzyme. METHODS: The therapeutic antioxidant enzyme superoxide dismutase (SOD) was covalently attached to the distal terminus of polyethylene glycol (PEG) polymer chains, located at the surface of lipid vesicles, to obtain SOD-enzymosomes. RESULTS: The in vivo fate of the optimized SOD-enzymosomes showed that SOD attachment at the end of the activated PEG slightly reduced the residence time of the liposome particles in the bloodstream after IV administration. The biodistribution studies showed that SOD-enzymosomes had a similar organ distribution profile to liposomes with SOD encapsulated in their aqueous interior (SOD-liposomes). SOD-enzymosomes showed earlier therapeutic activity than both SOD-liposomes and free SOD in rat adjuvant arthritis. SOD-enzymosomes, unlike SOD-liposomes, have a therapeutic effect, decreasing liver damage in a rat liver ischemia/reperfusion model. CONCLUSIONS: SOD-enzymosomes were shown to be a new and successful therapeutic approach to oxidative stress-associated inflammatory situations/diseases.

Pharmaceutical development and preclinical evaluation of a GMP-grade anti-inflammatory nanotherapy.

The present study describes the development of a good manufacturing practice (GMP)-grade liposomal nanotherapy containing prednisolone phosphate for the treatment of inflammatory diseases. After formulation design, GMP production was commenced which yielded consistent, stable liposomes sized 100nm±10nm, with a prednisolone phosphate (PLP) incorporation efficiency of 3%-5%. Pharmacokinetics and toxicokinetics of GMP-grade liposomal nanoparticles were evaluated in healthy rats, which were compared to daily and weekly administration of free prednisolone phosphate, revealing a long circulatory half-life with minimal side effects. Subsequently, non-invasive multimodal clinical imaging after liposomal nanotherapy's intravenous administration revealed anti-inflammatory effects on the vessel wall of atherosclerotic rabbits. The present program led to institutional review board approval for two clinical trials with patients with atherosclerosis. FROM THE CLINICAL EDITOR: In drug discovery, bringing production to industrial scale is an essential process. In this article the authors describe the development of an anti-inflammatory nanoparticle according to good manufacturing practice. As a result, this paves the way for translating laboratory studies to clinical trials in humans.

Prednisolone-containing liposomes accumulate in human atherosclerotic macrophages upon intravenous administration.

Drug delivery to atherosclerotic plaques via liposomal nanoparticles may improve therapeutic agents' risk-benefit ratios. Our paper details the first clinical studies of a liposomal nanoparticle encapsulating prednisolone (LN-PLP) in atherosclerosis. First, PLP's liposomal encapsulation improved its pharmacokinetic profile in humans (n=13) as attested by an increased plasma half-life of 63h (LN-PLP 1.5mg/kg). Second, intravenously infused LN-PLP appeared in 75% of the macrophages isolated from iliofemoral plaques of patients (n=14) referred for vascular surgery in a randomized, placebo-controlled trial. LN-PLP treatment did however not reduce arterial wall permeability or inflammation in patients with atherosclerotic disease (n=30), as assessed by multimodal imaging in a subsequent randomized, placebo-controlled study. In conclusion, we successfully delivered a long-circulating nanoparticle to atherosclerotic plaque macrophages in patients, whereas prednisolone accumulation in atherosclerotic lesions had no anti-inflammatory effect. Nonetheless, the present study provides guidance for development and imaging-assisted evaluation of future nanomedicine in atherosclerosis. FROM THE CLINICAL EDITOR: In this study, the authors undertook the first clinical trial using long-circulating liposomal nanoparticle encapsulating prednisolone in patients with atherosclerosis, based on previous animal studies. Despite little evidence of anti-inflammatory effect, the results have provided a starting point for future development of nanomedicine in cardiovascular diseases.

Synthesis and characterization of image-able polyvinyl alcohol microspheres for image-guided chemoembolization.

Therapeutic embolization of blood vessels is a minimally invasive, catheter-based procedure performed with solid or liquid emboli to treat bleeding, vascular malformations, and vascular tumors. Hepatocellular carcinoma (HCC) affects about half a million people per year. When unresectable, HCC is treated with embolization and local drug therapy by transarterial chemoembolization (TACE). For TACE, drug eluting beads (DC Bead(®)) may be used to occlude or reduce arterial blood supply and deliver chemotherapeutics locally to the tumor. Although this treatment has been shown to be safe and to improve patient survival, the procedure lacks imaging feedback regarding the location of embolic agent and drug coverage. To address this shortcoming, herein we report the synthesis and characterization of image-able drug eluting beads (iBeads) from the commercial DC Bead(®) product. Two different radiopaque beads were synthesized. In one approach, embolic beads were conjugated with 2,3,5-triiodobenzyl alcohol in the presence of 1,1'-carbonyldiimidazol to give iBead I. iBead II was synthesized with a similar approach but instead using a trimethylenediamine spacer and 2,3,5-triiodobenzoic acid. Doxorubicin was loaded into the iBeads II using a previously reported method. Size and shape of iBeads were evaluated using an upright microscope and their conspicuity assessed using a clinical CT and micro-CT. Bland and Dox-loaded iBeads II visualized with both clinical CT and microCT. Under microCT, individual bland and Dox loaded beads had a mean attenuation of 7904 ± 804 and 11,873.96 ± 706.12 HU, respectively. These iBeads have the potential to enhance image-guided TACE procedures by providing localization of embolic-particle and drug.

Triggered release of doxorubicin from temperature-sensitive poly(N-(2-hydroxypropyl)-methacrylamide mono/dilactate) grafted liposomes.

The objective of this study was to design temperature-sensitive liposomes with tunable release characteristics that release their content at an elevated temperature generated by high intensity focused ultrasound (HIFU) exposure. To this end, thermosensitive polymers of N-(2-hydroxypropyl)methacrylamide mono/dilactate of different molecular weights and composition with a cholesterol anchor (chol-pHPMAlac) were synthesized and grafted onto liposomes loaded with doxorubicin (DOX). The liposomes were incubated at different temperatures and their release kinetics were studied. A good correlation between the release-onset temperature of the liposomes and the cloud point (CP) of chol-pHPMAlac was found. However, release took place at significantly higher temperatures than the CP of chol-pHPMAlac, likely at the CP, the dehydration and thus hydrophobicity is insufficient to penetrate and permeabilize the liposomal membrane. Liposomes grafted with chol-pHPMAlac with a CP of 11.5 °C released 89% DOX within 5 min at 42 °C while for the liposomes grafted with a polymer with CP of 25.0 °C, a temperature of 52 °C was needed to obtain the same extent of DOX release. At a fixed copolymer composition, an increase in molecular weight from 6.5 to 14.5 kDa decreased the temperature at which DOX was released with a release-onset temperature from 52 to 42 °C. Liposomes grafted with 5% chol-pHPMAlac exhibited a rapid release to a temperature increase, while at a grafting density of 2 and 10%, the liposomes were less sensitive to an increase in temperature. Sequential release of DOX was obtained by mixing liposomes grafted with chol-pHPMAlac having different CPs. Chol-pHPMAlac grafted liposomes released DOX nearly quantitatively after pulsed wave HIFU. In conclusion, the release of DOX from liposomes grafted with thermosensitive polymers of N-(2-hydroxypropyl)methacrylamide mono/dilactate can be tuned to the characteristics and the grafting density of chol-pHPMAlac, making these liposomes attractive for local drug delivery using hyperthermia.

Hemocompatibility assessment of two siRNA nanocarrier formulations.

PURPOSE: Since the discovery of RNAi and its therapeutic potential, carrier systems have been developed to deliver small RNAs (particularly siRNA) for modulation of gene expression at the post-transcriptional level. An important factor determining the fate and usability of these systems in vivo is interaction with blood components, blood cells, and the immune system. In this study, a lipid-based and a polymer-based carrier system containing siRNA have been investigated in vitro in terms of their hemocompatibility. METHODS: The nanocomplexes studied were Angiplex, a targeted lipid-based system, and pHPMA-MPPM polyplex, a formulation based on a cationic polymer. siVEGFR-2 was encapsulated in both carriers and activation of platelets, coagulation, and complement cascade as well as induction of platelet aggregation were evaluated in vitro. RESULTS: Both systems had been shown before to cause significant silencing in vitro. Our findings indicated that pHPMA-MPPM polyplex triggered high platelet activation and aggregation although it did not stimulate coagulation substantially. Angiplex, on the other hand, provoked insignificant activation and aggregation of platelets and activated coagulation minimally. Complement system activation by Angiplex was in general low but stronger than pHPMA-MPPM polyplex. CONCLUSIONS: Taken together, these in vitro assays may help the selection of suitable carriers for systemic delivery of siRNA in early preclinical investigations and reduce the use of laboratory animals significantly.

Inhibition of tumor growth by targeted anti-EGFR/IGF-1R nanobullets depends on efficient blocking of cell survival pathways.

The clinical efficacy of epidermal growth factor receptor (EGFR)-targeted inhibitors is limited due to resistance mechanisms of the tumor such as activation of compensatory pathways. Crosstalk between EGFR and insulin-like growth factor 1 (IGF-1R) signaling has been frequently described to be involved in tumor proliferation and resistance. One of the attractive features of nanomedicines is the possibility to codeliver agents that inhibit different molecular targets in one nanocarrier system, thereby strengthening the antitumor effects of the individual agents. Additionally, exposure to healthy tissues and related unwanted side-effects can be reduced. To this end, we have recently developed anti-EGFR nanobody (Nb)-liposomes loaded with the anti-IGF-1R kinase inhibitor AG538, which showed promising antiproliferative effects in vitro. In the present study, we have further evaluated the potential of this dual-active nanomedicine in vitro and for the first time in vivo. As intended, the nanomedicine inhibited EGFR and IGF-1R signaling and subsequent activation of downstream cell proliferation and survival pathways. The degree of inhibition induced by the nanomedicine on a molecular level correlated with cytotoxicity in tumor cell proliferation assays and may even be predictive of the response to nanomedicine treatment in tumor xenograft models. Combination therapy with kinase inhibitor-loaded Nb-liposomes is therefore an appealing strategy for inhibiting the proliferation of tumors that are highly dependent on EGFR and IGF-1R signaling.

New insights into the HIFU-triggered release from polymeric micelles.

Continuous wave (CW), low frequency, high intensity focused ultrasound (HIFU) is a promising modality to trigger release of active compounds from polymeric micelles. The aim of the present study was to investigate whether high frequency CW as well as pulsed wave (PW) HIFU can induce the release of a hydrophobic agent from non-cross-linked (NCL) and core cross-linked (CCL) poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide-lactate] (mPEG-b-p(HPMAm-Lac(n))) micelles. It was shown that high frequency CW as well as PW HIFU was able to trigger the release (up to 85%) of a hydrophobic compound (i.e., nile red, NR) from NCL and CCL micelles. No changes in size distribution of the micelles after CW and PW HIFU exposure were observed and no degradation of polymer chain had occurred. We therefore hypothesize that the polymeric micelles are temporally destabilized upon HIFU exposure due to radiation force induced shear forces, leading to NR release on demand.

Evaluation of paclitaxel-loaded polymeric nanoparticles in 3D tumor model: impact of tumor stroma on penetration and efficacy.

Since tumor stroma poses as a barrier to achieve efficacy of nanomedicines, it is essential to evaluate nano-chemotherapeutics in stroma-mimicking 3D models that reliably predict their behavior regarding these hurdles limiting efficacy. In this study, we evaluated the effect of paclitaxel-loaded polymeric micelles (PTX-PMCs) and polymeric nanoparticles (PTX-PNPs) in a tumor stroma-mimicking 3D in vitro model. PTX-PMCs (77 nm) based on a amphiphilic block copolymer of mPEG-b-p(HPMAm-Bz) and PTX-PNPs (159 nm) based on poly(lactic-co-glycolic acid) were prepared, which had an encapsulation efficiency (EE%) of 81 ± 15% and 45 ± 8%, respectively. 3D homospheroids of mouse 4T1 breast cancer cells and heterospheroids of NIH3T3 fibroblasts and 4T1 (5:1 ratio) were prepared and characterized with high content two-photon microscopy and immunostaining. Data showed an induction of epithelial-mesenchymal transition (α-SMA) in both homo- and heterospheroids, while ECM (collagen) deposition only in heterospheroids. Two-photon imaging revealed that both fluorescently labeled PMCs and PNPs penetrated into the core of homospheroids and only PMCs penetrated into heterospheroids. Furthermore, PTX-PMCs, PTX-PNPs, and free PTX induced cytotoxicity in tumor cells and fibroblasts grown as monolayer, but these effects were substantially reduced in 3D models, in particular in heterospheroids. Gene expression analysis showed that heterospheroids had a significant increase of drug resistance markers (Bcl2, Abgc2) compared to 2D or 3D monocultures. Altogether, this study shows that the efficacy of nanotherapeutics is challenged by stroma-induced poor penetration and development of resistant phenotype. Therefore, this tumor stroma-mimicking 3D model can provide an excellent platform to study penetration and effects of nanotherapeutics before in vivo studies.

Platelets interact with CD169+ macrophages and cDC1 and enhance liposome-induced CD8+ T cell responses.

Historically platelets are mostly known for their crucial contribution to hemostasis, but there is growing understanding of their role in inflammation and immunity. The immunomodulatory role of platelets entails interaction with pathogens, but also with immune cells including macrophages and dendritic cells (DCs), to activate adaptive immune responses. In our previous work, we have demonstrated that splenic CD169+ macrophages scavenge liposomes and collaborate with conventional type 1 DCs (cDC1) to induce expansion of CD8+ T cells. Here, we show that platelets associate with liposomes and bind to DNGR-1/Clec9a and CD169/Siglec-1 receptors in vitro. In addition, platelets interacted with splenic CD169+ macrophages and cDC1 and further increased liposome internalization by cDC1. Most importantly, platelet depletion prior to liposomal immunization resulted in significantly diminished antigen-specific CD8+ T cell responses, but not germinal center B cell responses. Previously, complement C3 was shown to be essential for platelet-mediated CD8+ T cell activation during bacterial infection. However, after liposomal vaccination CD8+ T cell priming was not dependent on complement C3. While DCs from platelet-deficient mice exhibited unaltered maturation status, they did express lower levels of CCR7. In addition, in the absence of platelets, CCL5 plasma levels were significantly reduced. Overall, our findings demonstrate that platelets engage in a cross-talk with CD169+ macrophages and cDC1 and emphasize the importance of platelets in induction of CD8+ T cell responses in the context of liposomal vaccination.

Preclinical validation of a new hybrid molecule loaded in liposomes for melanoma management.

The aggressiveness of melanoma and lack of effective therapies incite the discovery of novel strategies. Recently, a new dual acting hybrid molecule (HM), combining a triazene and a ʟ-tyrosine analogue, was synthesized. HM was designed to specifically be activated by tyrosinase, the enzyme involved in melanin biosynthesis and overexpressed in melanoma. HM displayed remarkable superior antiproliferative activity towards various cancer cell lines compared with temozolomide (TMZ), a triazene drug in clinical use, that acts through DNA alkylation. In B16-F10 cells, HM induced a cell cycle arrest at phase G0/G1 with a 2.8-fold decrease in cell proliferation index. Also, compared to control cells, HM led to a concentration-dependent reduction in tyrosinase activity and increase in caspase 3/7 activity. To maximize the therapeutic performance of HM in vivo, its incorporation in long blood circulating liposomes, containing poly(ethylene glycol) (PEG) at their surface, was performed for passively targeting tumour sites. HM liposomes (LIP HM) exhibited high stability in biological fluids. Preclinical studies demonstrated its safety for systemic administration and in a subcutaneous murine melanoma model, significantly reduced tumour progression. In a metastatic murine melanoma model, a superior antitumour effect was also observed for mice receiving LIP HM, with markedly reduction of lung metastases compared to positive control group (TMZ). Biodistribution studies using 111In-labelled LIP HM demonstrated its ability for passively targeting tumour sites, thus correlating with the high therapeutic effect observed in the two experimental murine melanoma models. Overall, our proposed nanotherapeutic strategy was validated as an effective and safe alternative against melanoma.

Injectable liposomal docosahexaenoic acid alleviates atherosclerosis progression and enhances plaque stability.

Atherosclerosis is a chronic inflammatory vascular disease that is characterized by the accumulation of lipids and immune cells in plaques built up inside artery walls. Docosahexaenoic acid (DHA, 22:6n-3), an omega-3 polyunsaturated fatty acid (PUFA), which exerts anti-inflammatory and antioxidant properties, has long been purported to be of therapeutic benefit to atherosclerosis patients. However, large clinical trials have yielded inconsistent data, likely due to variations in the formulation, dosage, and bioavailability of DHA following oral intake. To fully exploit its potential therapeutic effects, we have developed an injectable liposomal DHA formulation intended for intravenous administration as a plaque-targeted nanomedicine. The liposomal formulation protects DHA against chemical degradation and increases its local concentration within atherosclerotic lesions. Mechanistically, DHA liposomes are readily phagocytosed by activated macrophages, exert potent anti-inflammatory and antioxidant effects, and inhibit foam cell formation. Upon intravenous administration, DHA liposomes accumulate preferentially in atherosclerotic lesional macrophages and promote polarization of macrophages towards an anti-inflammatory M2 phenotype, resulting in attenuation of atherosclerosis progression in both ApoE-/- and Ldlr-/- experimental models. Plaque composition analysis demonstrates that liposomal DHA inhibits macrophage infiltration, reduces lipid deposition, and increases collagen content, thus improving the stability of atherosclerotic plaques against rupture. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) further reveals that DHA liposomes can partly restore the complex lipid profile of the plaques to that of early-stage plaques. In conclusion, DHA liposomes offer a promising approach for applying DHA to stabilize atherosclerotic plaques and attenuate atherosclerosis progression, thereby preventing atherosclerosis-related cardiovascular events.