Samuli Hirsjärvi

Passive and active tumour targeting with nanocarriers.

Nanocarriers can penetrate the tumour vasculature through its leaky endothelium and, in this way, accumulate in several solid tumours. This is called the enhanced permeation and retention (EPR) effect. Together with nanocarriers whose surface is tailored for prolonged blood circulation times, the concept is referred to as passive targeting. Targeting ligands, which bind to specific receptors on the tumour cells and endothelium, can be attached on the nanocarrier surface. This active targeting increases the selectivity of the delivery of drugs. Passive and active drug targeting with nanocarriers to tumours reduce toxic side-effects, increase efficacy, and enhance delivery of poorly soluble or sensitive therapeutic molecules. In this review, currently studied and used passive and active targeting strategies in cancer therapy are presented.

Influence of size, surface coating and fine chemical composition on the in vitro reactivity and in vivo biodistribution of lipid nanocapsules versus lipid nanoemulsions in cancer models

UNLABELLED Lipid nanocapsules (LNCs) and lipid nanoemulsions (LNEs) are biomimetic synthetic nanocarriers. Their in vitro and in vivo performance was evaluated as a function of their size (25, 50 and 100 nm) and the surface PEG chain length. Analysis methods included complement activation test, particle uptake in macrophage and HEK293(β3) cells and biodistribution studies with tumor-grafted mice by fluorescence imaging. A particular attention was paid to keep the concentration of each nanocarrier and to the amount of fluorescent dye in comparable conditions between the in vitro and in vivo studies. Under these conditions, no significant differences were found among the three tested particle sizes and the two nanocarrier types. Longer PEG chains on the LNE surface provided better stealth properties, whereas PEG modification on the LNC formulations inhibited the production of stable nanocarriers. Passive accumulation of LNCs and LNEs in different tumor types depended on the degree of tumor vascularization. FROM THE CLINICAL EDITOR This study of lipid nanocapsules and lipid nanoemulsions compares their vitro and in vivo performance as a function of size and surface PEG chain length, demonstrating no significant difference among the tested particle sizes. Longer PEG chains on the LNE surface provided better stealth properties, whereas PEG modification on the LNC formulations inhibited the production of stable nanocarriers.

FRET Imaging Approaches for in Vitro and in Vivo Characterization of Synthetic Lipid Nanoparticles

DiI and DiD, two fluorophores able to interact by FRET (Förster resonance energy transfer), were coencapsulated in the core of lipid nanocapsules (LNCs) and nanoemulsions (LNEs), lipophilic reservoirs for the delivery of drugs. The ability of FRET imaging to provide information on the kinetics of dissociation of the nanoparticles in the presence of bovine serum albumin (BSA) or whole serum, or after incubation with cancer cells, and after systemic administration in tumor-bearing mice, was studied. Both microscopic and macroscopic imaging was performed to determine the behavior of the nanostructures in a biological environment. When 2 mg/mL FRET LNEs or LNCs were dispersed in buffer, in the presence of unloaded nanoparticles, BSA, or in whole serum, the presence of serum was the most active in destroying the particles. This occurred immediately with a diminution of 20% of FRET, then slowly, ending up with still 30% intact nanoparticles at 24 h. LNCs were internalized rapidly in cultured cells with the FRET signal decreasing within the first minutes of incubation, and then a plateau was reached and LNCs remained intact during 3 h. In contrast, LNEs were poorly internalized and were rapidly dissociated after internalization. Following their iv injection, LNCs appeared very stable in subcutaneous tumors implanted in mice. Intact particles were found using microscopic FRET determination on tumor sections 24 h after injection, that correlated well with the 8% calculated noninvasively on live animals. FRET investigations showed the potential to determine valid and reliable information about in vitro and in vivo behavior of nanoparticles.

Layer-by-layer surface modification of lipid nanocapsules

Lipid nanocapsules (LNCs) were modified by adsorbing sequentially dextran sulfate (DS) and chitosan (CS) on their surface by the layer-by-layer (LBL) approach. Tangential flow filtration (TFF) was used in intermediate purifications of the LNC dispersion during the LBL process. The surface modification was based on electrostatic interactions between the coating polyelectrolytes (PEs) and the LNCs. Therefore, a cationic surfactant, lipochitosan (LC), was synthesised by coupling stearic anhydride on chitosan, and the surface of LNCs was first modified by this LC by the post-insertion technique. The PEs could be successfully adsorbed on the LNC surface as verified by alternating zeta potential and increase in size. To present a therapeutic application, fondaparinux sodium (FP), a heparin-like synthetic pentasaccharide, was introduced on the LNC surface instead of DS.

Effect of particle size on the biodistribution of lipid nanocapsules: Comparison between nuclear and fluorescence imaging and counting

In vivo biodistribution of nanoparticles depends on several physicochemical parameters such as size. After intravenous injection of 25, 50 and 100 nm lipid nanocapsules (LNC) in nude mice bearing HEK293(β3) tumour xenografts, biodistribution was evaluated by γ-scintigraphy and by γ-counting. The small LNC 25 nm disappeared faster than the larger LNC 50 and 100 nm from the blood circulation due to faster elimination and wider tissue distribution. At 24h, biodistribution profiles of all these LNC were similar. Low LNC quantities were found in this weak EPR (enhanced permeability and retention) tumour regardless the particle size. Co-injected 50 nm fluorescent DiD-LNC and (99m)Tc-LNC allowed direct comparison of biodistribution as evaluated by the two methods. Optical imaging underestimated LNC quantity especially in dark-colored organs that were observed to capture extensive quantities of the particles by γ-counting (i.e. liver, spleen, and kidney).

Surface modification of lipid nanocapsules with polysaccharides: from physicochemical characteristics to in vivo aspects.

Attaching polysaccharides to the surface of nanoparticles offers the possibility of modifying the physicochemical and biological properties of the core particles. The surface of lipid nanocapsules (LNCs) was modified by post-insertion of amphiphilic lipochitosan (LC) or lipodextran (LD). Modelling of these LNCs by the drop tensiometer technique revealed that the positively charged LC made the LNC surface more rigid, whereas the neutral, higher M(W) LD had no effect on the surface elasticity. Both LNC-LC and LNC-LD activated the complement system more than the blank LNC, thus suggesting increased capture by the mononuclear phagocyte system. In vitro, the positively charged LNC-LC were more efficiently bound by the model HEK293(β3) cells compared to LNC and LNC-LD. Finally, it was observed that neither LC nor LD changed the in vivo biodistribution properties of LNCs in mice. These polysaccharide-coated LNCs, especially LNC-LC, are promising templates for targeting ligands (e.g. peptides, proteins) or therapeutic molecules (e.g. siRNA).

An MRI-based classification scheme to predict passive access of 5 to 50-nm large nanoparticles to tumors

Nanoparticles are useful tools in oncology because of their capacity to passively accumulate in tumors in particular via the enhanced permeability and retention (EPR) effect. However, the importance and reliability of this effect remains controversial and quite often unpredictable. In this preclinical study, we used optical imaging to detect the accumulation of three types of fluorescent nanoparticles in eight different subcutaneous and orthotopic tumor models, and dynamic contrast-enhanced and vessel size index Magnetic Resonance Imaging (MRI) to measure the functional parameters of these tumors. The results demonstrate that the permeability and blood volume fraction determined by MRI are useful parameters for predicting the capacity of a tumor to accumulate nanoparticles. Translated to a clinical situation, this strategy could help anticipate the EPR effect of a particular tumor and thus its accessibility to nanomedicines.

Evaluation of surface deformability of lipid nanocapsules by drop tensiometer technique, and its experimental assessment by dialysis and tangential flow filtration.

Deformability of nanoparticles might affect their behaviour at biological interfaces. Lipid nanocapsules (LNCs) are semi-solid particles resembling a hybrid of polymer nanoparticles and liposomes. Deformability of LNCs of different sizes was modelled by drop tensiometer technique. Two purification methods, dialysis and tangential flow filtration (TFF), were applied to study experimental behaviour and deformability of LNCs in order to evaluate if these properties contributed to membrane passing. Rheological parameters obtained from the drop tensiometer analysis suggested decreasing surface deformability of LNCs with increase in diameter. Dialysis results showed that up to 10% of LNCs can be lost during the process (e.g. membrane accumulation) but no clear evidence of the membrane passing was observed. Instead, LNCs with initial size and size distribution could be found in the TFF filtrate although molecular weight cut-off (MWCO) of the membrane used was smaller than the LNC diameter.

Tumour targeting of lipid nanocapsules grafted with cRGD peptides.

Combining targeting to therapy remains a major challenge in cancer treatment. To address this subject, the surface of lipid nanocapsules (LNC) was modified by grafting cRGD peptides, which are known to be recognised by αvβ3 integrins expressed by tumour endothelium and cancer cells. Applicability of this LNC-cRGD in tumour targeting was first assessed in vitro by the use of U87MG glioma cells. Biodistribution and tumour accumulation of radiolabelled LNC-cRGD in vivo were then evaluated in mice bearing the same subcutaneous xenograft. Flow cytometry and confocal microscopy results revealed that the cRGD grafting improved binding and internalisation compared to negative control LNC-cRAD and blank LNC. The peptide-grafted LNC remained in the blood circulation up to 3h with reduced capture by the RES organs. Tumour accumulation of LNC-cRGD with respect to LNC-cRAD was significantly higher at 1-3h. These results show that cRGD grafted to LNC has created a promising tumour-targetable nanocarrier that could be used in cancer treatment.

Chapter 8.3:Drug Delivery Strategies: Lipid Nanocapsules

Over the past few decades, the development of nanomedicines has been significant with the discovery of new drugs, new targets and also new drug delivery systems. The link between nanotechnological development and medicine can provide medical and pharmaceutical benefits, particularly in the field of ...