Surface Of Lipid Nanocapsules Research Articles

Glioblastoma-targeted, local and sustained drug delivery system based on an unconventional lipid nanocapsule hydrogel

The objective of this work was to develop an implantable therapeutic hydrogel that will ensure continuity in treatment between surgery and radiochemotherapy for patients with glioblastoma (GBM). A hydrogel of self-associated gemcitabine-loaded lipid nanocapsules (LNC) has shown therapeutic efficacy in vivo in murine GBM resection models. To improve the targeting of GBM cells, the NFL-TBS.40–63 peptide (NFL), was associated with LNC. The LNC-based hydrogels were formulated with the NFL. The peptide was totally and instantaneously adsorbed at the LNC surface, without modifying the hydrogel mechanical properties, and remained adsorbed to the LNC surface after the hydrogel dissolution. In vitro studies on GBM cell lines showed a faster internalization of the LNC and enhanced cytotoxicity, in the presence of NFL. Finally, in vivo studies in the murine GBM resection model proved that the gemcitabine-loaded LNC with adsorbed NFL could target the non-resected GBM cells and significantly delay or even inhibit the apparition of recurrences.

Lipid nanocapsules for the nose-to-brain delivery of the anti-inflammatory bioactive lipid PGD2-G

Here, prostaglandin D2-glycerol ester (PGD2-G) was selected to target neuroinflammation. As PGD2-G is reported to have a short plasmatic half-life, we propose to use lipid nanocapsules (LNC) as vehicle to safely transport PGD2-G to the central nervous system (CNS). PGD2-G-loaded LNC (PGD2-G-LNC) reduced pro-inflammatory cytokine expression in activated microglial cells, even so after crossing a primary olfactory cell monolayer. A single nasal administration of PGD2-G-LNC in lipopolysaccharide (LPS)-treated mice reduced pro-inflammatory cytokine expression in the olfactory bulb. Coating LNC's surface with a cell-penetrating peptide, transactivator of transcription (TAT), increased its accumulation in the brain. Although TAT-coated PGD2-G-LNC modestly exerted its anti-inflammatory effect in a mouse model of multiple sclerosis similar to free PGD2-G after nasal administration, TAT-coated LNC surprisingly reduced the expression of pro-inflammatory chemokines in the CNS. These data propose LNC as an interesting drug delivery tool and TAT-coated PGD2-G-LNC remains a good candidate, in need of further work.

PH-Responsive Lipid Nanocapsules: A Promising Strategy for Improved Resistant Melanoma Cell Internalization.

Despite significant advances in melanoma therapy, low response rates and multidrug resistance (MDR) have been described, reducing the anticancer efficacy of the administered molecules. Among the causes to explain these resistances, the decreased intratumoral pH is known to potentiate MDR and to reduce the sensitivity to anticancer molecules. Nanomedicines have been widely exploited as the carriers of MDR reversing molecules. Lipid nanocapsules (LNC) are nanoparticles that have already demonstrated their ability to improve cancer treatment. Here, LNC were modified with novel copolymers that combine N-vinylpyrrolidone (NVP) to impart stealth properties and vinyl imidazole (Vim), providing pH-responsive ability to address classical chemoresistance by improving tumor cell entry. These copolymers could be post-inserted at the LNC surface, leading to the property of going from neutral charge under physiological pH to positive charge under acidic conditions. LNC modified with polymer P5 (C18H37-P(NVP21-co-Vim15)) showed in vitro pH-responsive properties characterized by an enhanced cellular uptake under acidic conditions. Moreover, P5 surface modification led to an increased biological effect by protecting the nanocarrier from opsonization by complement activation. These data suggest that pH-sensitive LNC responds to what is expected from a promising nanocarrier to target metastatic melanoma.

The origin of neural stem cells impacts their interactions with targeted-lipid nanocapsules: Potential role of plasma membrane lipid composition and fluidity

The adsorption of a peptide (NFL-TBS.40–63 peptide (NFL)) known to induce neural stem cells (NSC) differentiation in vitro, at the surface of lipid nanocapsules (LNC) provides a targeting drug delivery system (NFL-LNC) that penetrates subventricular zone-neural stem cells (SVZ-NSC) but not central canal-NSC (CC-NSC). We hypothesized preferential interactions could explaine, at least partially, the different properties of SVZ- and CC-NSC plasma membranes. The objective of this work was to compare SVZ- and CC-NSC plasma membrane lipid composition, fluidity and permeability. Plasma membranes of SVZ- and CC-NSC were isolated and analyzed by LC-MS for their lipid content. Membrane fluidity was evaluated by measuring the generalized polarization (GP) of Laurdan and membrane permeability by fluorescent dextran penetration. Liposomes with different lipid compositions and steady state fluidities were prepared. ΔGP was measured after incubation with NFL-LNC. A significantly higher proportion of cholesterol, ceramides, sphingomyelins, phosphatidylethanolamines and a lower proportion of phosphatidylcholines and sulfatides were observed in SVZ- compared to CC-NSC. Fluidity, probably more than lipid composition, drove NFL-LNC and NSC interactions, and SVZ-NSC were more sensitive to NFL permeabilization than CC-NSC. We demonstrated that NSC membrane lipid composition and fluidity depended of NSC origin and that these features could play a role in the specific interactions with NFL-LNC.

Lipid nanocapsules functionalized with polyethyleneimine for plasmid DNA and drug co-delivery and cell imaging.

The paper reports on the preparation of lipid nanocapsules (LNCs) functionalized with poly(ethyleneimine) (PEI) moieties and their successful use as drug and gene delivery systems. The cationic LNCs were produced by a phase inversion process with a nominal size of 25 nm and subsequently modified with PEI chains using a transacylation reaction. The functionalization process allowed good control over the nanoscale particle size (26.2 ± 3.9 nm) with monodisperse size characteristics (PI < 0.2) and positive surface charge up to +18.7 mV. The PEI-modified LNCs (LNC25-T) displayed good buffering capacity. Moreover, the cationic LNC25-T were able to condense DNA and form complexes via electrostatic interactions in a typical weight ratio-dependent relationship. It was found that the mean diameter of LNC25-T/pDNA complexes increased to ∼40-50 nm with the LNC25-T/pDNA ratio from 1 to 500. Gel electrophoresis and cell viability experiments showed that the LNC25-T/pDNA complexes had high stability with no cytotoxicity due to the anchored PEI polymers on the surface of LNCs. Finally, the transfection efficiency of the LNC25-T/pDNA complexes was studied and evaluated on HEK cell lines in comparison with free PEI/pDNA polyplexes. The combination of cationic LNCs with pDNA exhibited more than a 2.8-fold increase in transfection efficiency compared to the standard free PEI/pDNA polyplexes at the same PEI concentrations. Moreover, we have demonstrated that LNC25-T/pDNA loaded with a hydrophobic drug, paclitaxel, showed high drug efficacy. The high transfection efficiency combined with the potential of simultaneous co-delivery of hydrophobic drugs, relatively small size of LNC25-T/pDNA complexes, and fluorescence imaging can be crucial for gene therapy, as small particle sizes may be more favorable for in vivo studies.

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.

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 MW 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).

Brain tumour targeting strategies via coated ferrociphenol lipid nanocapsules

In this study, a new active targeting strategy to favour ferrociphenol (FcdiOH) internalisation into brain tumour cells was developed by the use of lipid nanocapsules (LNCs) coated with a cell-internalising peptide (NFL-TBS.40–63 peptide) that interacts with tubulin-binding sites. In comparison, OX26 murine monoclonal antibodies (OX26-MAb) targeting transferrin receptors were also inserted onto the LNC surface. The incorporation of OX26 or peptide did not influence the in vitro antiproliferative effect of FcdiOH–LNCs on the 9L cells since their IC50 values were found in the same range. In vivo, intracerebral administration of OX26–FcdiOH–LNCs or peptide–FcdiOH–LNCs by convection enhanced delivery did not enhance the animal median survival time in comparison with untreated rats (25days). Interestingly, intra-carotid treatment with peptide–FcdiOH–LNCs led to an ameliorated survival time of treated rats with the presence of animals surviving until days 35, 40 and 44. Such results were not obtained with OX26-MAbs, demonstrating the benefit of NFL-TBS.40–63 peptide as an active ligand for peripheral drug delivery to the brain tumours.

Treatment of 9L Gliosarcoma in Rats by Ferrociphenol-Loaded Lipid Nanocapsules Based on a Passive Targeting Strategy via the EPR Effect

To study a passive targeting strategy, via the enhanced permeability and retention effect following systemic administration of lipid nanocapsules (LNCs) loaded with ferrociphenol, FcdiOH. Long chains of polyethylene glycol (DSPE-mPEG2000) were incorporated onto the surface of LNCs by post-insertion technique. Stealth properties of LNCs were investigated by in vitro complement consumption and macrophage uptake, and in vivo pharmacokinetics in healthy rats. Antitumour effect of FcdiOH-loaded LNCs was evaluated in subcutaneous and intracranial 9L gliosarcoma rat models. LNCs and DSPE-mPEG2000-LNCs presented low complement activation and weak macrophage uptake. DSPE-mPEG2000-LNCs exhibited prolonged half-life and extended area under the curve in healthy rats. In a subcutaneous gliosarcoma model, a single intravenous injection of FcdiOH-LNCs (400 μL, 2.4 mg/rat) considerably inhibited tumour growth when compared to the control. DSPE-mPEG2000-FcdiOH-LNCs exhibited a strong antitumour effect by nearly eradicating the tumour by the end of the study. In intracranial gliosarcoma model, treatment with DSPE-mPEG2000-FcdiOH-LNCs and FcdiOH-LNCs statistically improved median survival time (28 and 27.5 days, respectively) compared to the control (25 days). These results demonstrate the interesting perspectives for the systemic treatment of glioma thanks to bio-organometallic chemotherapy via lipid nanocapsules.

Development and characterization of immuno-nanocarriers targeting the cancer stem cell marker AC133

In the context of targeted therapy, we addressed the possibility of developing a drug delivery nanocarrier capable to specifically reach cancer cells that express the most prominent marker associated with cancer stem cell (CSC) phenotype, AC133. For this purpose, 100 nm lipid nanocapsules (LNCs) were functionalized with a monoclonal antibody (mAb) directed against AC133 according to two distinct methods: firstly, post-insertion within 100 nm LNCs of a lipid poly(ethylene glycol) functionalized with reactive-sulfhydryl maleimide groups (DSPE-PEG 2000-maleimide) followed by thiolated mAb coupling, and, secondly, creation of a thiolated lipo-immunoglobulin between DSPE-PEG 2000-maleimide and AC133, then post-inserted within LNCs. Due to the reduced number of purification steps, lower amounts of DSPE-PEG 2000-maleimide that were necessary as well as lower number of free maleimide functions present onto the surface of immuno-LNC, the second method was found to be more appropriate. Thus, 126 nm AC133-LNC with a zeta potential of −22 mV while keeping a narrow distribution were developed. Use of the IgG1κ isotype control-immunoglobulins produced similar control IgG1-LNCs. Micro-Bradford colorimetric assay indicated a fixation of about 40 immunoglobulins per LNC. Use of human Caco-2 cells that constitutively express AC133 (Caco-2-AC133 high) allowed addressing the behavior of the newly functionalized immuno-LNCs. siRNA knockown strategy permitted to obtain Caco-2-AC133 low for comparison. Immunofluorescence-combined flow cytometry analysis demonstrated that the epitope-recognition function of AC133 antibody was preserved when present on immuno-LNCs. Although grafting of immunoglobulins onto the surface of LNCs repressed their internalization within Caco-2 cells as evaluated by flow cytometry, AC133-specific cellular binding was obtained with AC133-LNC as assessed by computer-assisted fluorescence microscopy. In conclusion, interest of AC133-LNCs as niche carriers is discussed toward the development of CSC targeted chemo- or radio-nanomedicines.

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.

Abstract A60: Targeting brain tumor stem cells

Abstract The brain tumor stem-cell (BTSC) hypothesis proposes with acquired self-renewal properties establish a brain tumorigenic-behavior (1). Thus, failure to cure brain cancer has been attributed to the fact that typical cytotoxic therapies target rapidly proliferating brain tumor cells, while sparing the BTSC compartment, which has a low proliferation rate and high tumorigenic potential. Taking advantage of the recent development of nanotechnologies that may offer innovative drug-delivery systems for improved efficacy, specificity and biological safety (2), the aim of our work is to develop nanoparticle-based treatments targeting BTSCs. The first part of our work represents a comprehensive study of the aggressiveness of the BTSC was shown to be associated with the expression of the neural precursor cell marker AC133. Hence, the percentage of AC133 expressing glioma cells is increased after maintenance or exposure to moderately low oxygen levels (3). Our data indicate that cells maintained at 3% oxygen express more AC133 and are more aggressive after in vivo xenograft than cells cultured at 21% oxygen. Thus, oxygen pressure represents a major parameter in the expression of the AC133 molecule but is also important in aggressiveness of glioma cells. The mechanism that links hypoxia, AC133 expression and the tumorigenic behavior of BTSC has been studied. Our results, including CoCl2 induction and shRNA knockdown, suggest a role for HIF-1α in the regulation of AC133 in glioblastoma cells. The second part of our work, immunonanoparticules (ILNC) that target AC133 were synthesized by conjugation of thiolated AC133 or IgG1 isotype control immunoglobulins to lipid nanocapsules (LNC) (4) by linkage through incorporation of lipid PEG(2000) functionalized with reactive-sulfhydryl maleimide groups (DSPE-PEG(2000)-maleimide) on the surface of LNC by using a post-insertion procedure (5). Our results indicate a fixation yield of antibodies on LNC varying from 6 to 60% representing 70 antibodies per LNC. To evaluate the selectivity of immunonanoparticles binding and resulting uptake by cells a second in vitro model has been developed. Human Caco-2 cells, previously described to express AC133, were used and exposed to siRNA targeting the CD133 gene to dispose of two types of Caco-2 cells: Caco-2-AC133high and Caco-2-AC133low. We are now testing ILNC uptake in our Caco-2 cell model. We will test if ILNC are able to bind to AC133 epitope on Caco-2 cells and if they are able to be internalized in cells. Accumulated results must allow us to extend our knowledge on glioma aggressiveness and to adapt our vectors to resolve the BTSC targeting problem and their death. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A60.

In vivo evaluation of intracellular drug-nanocarriers infused into intracranial tumours by convection-enhanced delivery: distribution and radiosensitisation efficacy

The objective of the present study was to investigate the interest of convection-enhanced delivery (CED) for the administration of a nanocarrier-based radiosensitizing chemotherapy in the rat brain. Pursuing on newly developed lipid nanocapsules (LNC) that can be internalised within brain tumour cells, we studied their intracerebral distribution when labelled with fluorescent Nile red (NR). As paclitaxel (Px) represents an interesting radiosensitiser, we also evaluated the potential radiosensitising effects of Px-loaded LNC administered through CED in the 9L intracranial rat glioblastoma model. The distribution study demonstrated that CED injection of NR-loaded LNC (NR-LNC) improved significantly the volume of distribution of NR when matched with simple injection (by about 150 fold). It also reveals that the LNC perfusion of a whole tumour forming area inside the CNS (6 days after implantation of 10(3) 9L cells) is achievable through CED injection, whilst preserving the ability of LNC to reach the intracellular space of encountered tumour cells. Having established an animal model of encephalic irradiation close to the clinic (18 Gray in three fractions of six Gray at days 8, 11 and 14 after 9L cell implantation) we proved the feasibility of the combination of CED for the administration of drug-loaded LNC with external beam therapy. Although a single CED injection of Px-LNC at low Px dose (375 mug/kg of bodyweight) gave the best median survival (twice that of untreated controls), it underlines the need for optimisation. Hence, the possibility of grafting recognition moieties onto the LNC surface combined to their biocompatibility must be beneficial.

New lipid nanocapsules exhibit sustained release properties for amiodarone

Amiodarone is widely used in heart diseases but also provokes severe adverse effects due to its accumulation in other tissues than the heart. In order to circumvent side effects colloidal drug carriers have been designed to deliver the drug specifically to the site of action. Many preparation methods have been described and most have been reported to involve a high initial drug loss when introduced in an aqueous environment. Lipid nanocapsules (LNC) were prepared by a new phase inversion procedure and characterized in terms of size, surface potential, encapsulation efficiency, and drug release pattern. The encapsulation rate was varying between 92 and 94%. LNC did not display a distinct initial burst effect while the drug release of amiodarone can be prolonged over a significant period. Acceptor phase interfaces such as liposomes or blank LNC were applied to the release medium to enable a drug release to larger extents. The release was triggered by the pH of the release medium showing a faster release for lower pH; t 50% values vary from 25.6 h (pH 2) to 236.3 h (pH 7.4). Moreover, LNC were prepared of different sizes (24.7±2.0 to 102.5±0.9 nm) showing only slight influences on their drug release profiles. It was concluded that the LNC surface is able to retain amphiphilic drugs. Such properties could allow drug delivery to the site of action without high initial drug loss.