PLA-PEG Nanoparticles Improve the Anti-Inflammatory Effect of Rosiglitazone on Macrophages by Enhancing Drug Uptake Compared to Free Rosiglitazone.

Among cardiovascular diseases, atherosclerosis remains the first cause of death in the United States of America and Europe, as it leads to myocardial infarction or stroke. The high prevalence of heart diseases is due to the difficulty in diagnosing atherosclerosis, since it can develop for decades before symptoms occur, and to the complexity of the treatment since targets are also important components of the host defenses. The antidiabetics thiazolidinediones, among which is rosiglitazone (RSG), have demonstrated anti-atherosclerotic effect in animal models, and are therefore promising candidates for the improvement of atherosclerosis management. Nevertheless, their administration is hindered by the insurgence of severe side effects. To overcome this limitation, rosiglitazone has been encapsulated into polymeric nanoparticles, which permit efficient delivery to its nuclear target, and selective delivery to the site of action, allowing the reduction of unwanted effects. In the present work, we describe nanoparticle formulation using polylactic acid (PLA) coupled to polyethylene glycol (PEG), their characterization, and their behavior on RAW264.7 macrophages, an important target in atherosclerosis treatment. RSG nanocarriers showed no toxicity on cells at all concentrations tested, an anti-inflammatory effect in a dose-dependent manner, up to 5 times more efficient than the free molecule, and an increased RSG uptake which is consistent with the effect shown. These biodegradable nanoparticles represent a valid tool to be further investigated for the treatment of atherosclerosis.

Ultrasound-induced mild hyperthermia improves the anticancer efficacy of both Taxol® and paclitaxel-loaded nanocapsules.

We study the influence of ultrasound on paclitaxel-loaded nanocapsules in vitro and in vivo. These nanocapsules possess a shell of poly(dl-lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) and a liquid core of perfluorooctyl bromide (PFOB). In vitro experiments show that mechanical effects such as cavitation are negligible for nanocapsules due to their small size and thick and rigid shell. As the mechanical effects were unable to increase paclitaxel delivery, we focused on the thermal effects of ultrasound in the in vivo studies. A focused ultrasound sequence was therefore optimized in vivo under magnetic resonance imaging guidance to obtain localized mild hyperthermia with high acoustic pressure. Ultrasound-induced mild hyperthermia (41-43°C) was then tested in vivo in a subcutaneous CT-26 colon cancer murine model. As hyperthermia is applied, an inhibition of tumor growth for both paclitaxel-loaded nanocapsules and the commercial formulation of paclitaxel, namely Taxol® have been observed (p<0.05). Ultrasound-induced mild hyperthermia at high acoustic pressure appears as an interesting strategy to enhance cytotoxic efficacy locally.

Paclitaxel-loaded PEGylated nanocapsules of perfluorooctyl bromide as theranostic agents.

We optimize the encapsulation of paclitaxel (PTX) into nanocapsules made of a shell of poly(lactide-co-glycolide)-polyethylene glycol and a core of perfluorooctyl bromide (PFOB) to serve as theranostic agents. Two main challenges were met: keeping the imaging moiety (PFOB) encapsulated while loading the polymer shell with a hydrophobic drug very prone to crystallization. Encapsulation is performed by a modified emulsion-evaporation method leading to 120nm diameter nanocapsules with a drug loading compatible with tumor treatment. The optimized formulation tested in vitro on CT-26 colon cancer cells yields a similar IC50 as the generic Taxol® formulation. In vivo, 19F-MRI shows that PTX encapsulation does not modify the ability of nanocapsules to accumulate passively in CT-26 tumors in mice by the enhanced permeation and retention (EPR) effect. This accumulation leads to a promising and statistically significant twofold reduction in tumor growth as compared with negative control and generic Taxol® group. Altogether these results advocate for an interesting potential of these paclitaxel-loaded theranostic agents.

Pharmacokinetics, biodistribution and metabolism of squalenoyl adenosine nanoparticles in mice using dual radio-labeling and radio-HPLC analysis.

Adenosine is a pleiotropic endogenous nucleoside with potential neuroprotective pharmacological activity. However, clinical use of adenosine is hampered by its extremely fast metabolization. To overcome this limitation, we recently developed a new squalenoyl nanomedicine of adenosine [Squalenoyl-Adenosine (SQAd)] by covalent linkage of this nucleoside to the squalene, a natural lipid. The resulting nanoassemblies (NAs) displayed a dramatic pharmacological activity both in cerebral ischemia and spinal cord injury pre-clinical models. The aim of the present study was to investigate the plasma profile and tissue distribution of SQAd NAs using both Squalenoyl-[(3)H]-Adenosine NAs and [(14)C]-Squalenoyl-Adenosine NAs as respective tracers of adenosine and squalene moieties of the SQAd bioconjugate. This study was completed by radio-HPLC analysis allowing to determine the metabolization profile of SQAd. We report here that SQAd NAs allowed a sustained circulation of adenosine under its prodrug form (SQAd) for at least 1h after intravenous administration, when free adenosine was metabolized within seconds after injection. Moreover, the squalenoylation of adenosine and its formulation as NAs also significantly modified biodistribution, as SQAd NAs were mainly captured by the liver and spleen, allowing a significant release of adenosine in the liver parenchyma. Altogether, these results suggest that SQAd NAs provided a reservoir of adenosine into the bloodstream which may explain the previously observed neuroprotective efficacy of SQAd NAs against cerebral ischemia and spinal cord injury.

Formulation and pharmacokinetics of thermosensitive stealth® liposomes encapsulating 5-Fluorouracil.

PURPOSE: We optimize the encapsulation and investigate the pharmacokinetics of 5-Fluorouracil (5-FU) delivered by thermosensitive stealth(®) liposomes (TSLs) designed to trigger drug release upon hyperthermia using focused ultrasound (FUS). METHODS: 5-FU was encapsulated into liposomes made of 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine/cholesterol/1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG2000 either as a free molecule or complexed with copper-polyethylenimine. Heat-triggered drug release was evaluated using either a water bath or FUS. Formulation cytotoxicity was assessed on HT-29 cell line by MTS assay. Pharmacokinetics and biodistribution of 5-FU were evaluated in HT-29-tumor bearing mice. RESULTS: 5-FU was easily encapsulated using the lipid hydration method (encapsulation efficacy of 13%) but poorly retained upon dilution. 5-FU complexation with copper-polyethylenimine improved 5-FU retention into liposomes and allowed to obtain an encapsulation efficacy of 37%. At 42°C, heat-triggered 5-FU release from TSLs was 63% using a water bath and 68% using FUS, within 10 min, whereas it remained below 20% for the non-thermosensitive formulation. The MTS assay revealed that formulation toxicity arose from 5-FU and not from the excipients. In addition, 5-FU complex encapsulation into TSLs induces a reduction of the IC50 from 115 down to 49 µM. Pharmacokinetics reveals a longer circulation of encapsulated 5-FU and a more important body exposure, although tumor passive targeting is not significantly higher than free 5-FU. CONCLUSIONS: Complexation of 5-FU with copper-polyethylenimine appears an interesting strategy to improve 5-FU retention into TSLs in vitro and in vivo. TSLs allow heat-triggered release of the drug within 10 min at 42°C, a reasonable time for future in vivo experiments.

Squalenoyl adenosine nanoparticles provide neuroprotection after stroke and spinal cord injury.

There is an urgent need to develop new therapeutic approaches for the treatment of severe neurological trauma, such as stroke and spinal cord injuries. However, many drugs with potential neuropharmacological activity, such as adenosine, are inefficient upon systemic administration because of their fast metabolization and rapid clearance from the bloodstream. Here, we show that conjugation of adenosine to the lipid squalene and the subsequent formation of nanoassemblies allows prolonged circulation of this nucleoside, providing neuroprotection in mouse stroke and rat spinal cord injury models. The animals receiving systemic administration of squalenoyl adenosine nanoassemblies showed a significant improvement of their neurologic deficit score in the case of cerebral ischaemia, and an early motor recovery of the hindlimbs in the case of spinal cord injury. Moreover, in vitro and in vivo studies demonstrated that the nanoassemblies were able to extend adenosine circulation and its interaction with the neurovascular unit. This Article shows, for the first time, that a hydrophilic and rapidly metabolized molecule such as adenosine may become pharmacologically efficient owing to a single conjugation with the lipid squalene.

Stabilization and cellular delivery of chitosan-polyphosphate nanoparticles by incorporation of iron.

Chitosan (CS) nanoparticles are typically obtained by complexation with tripolyphosphate (TPP) ions, or more recently using triphosphate group-containing drugs such as adenosine triphosphate (ATP). ATP is an active molecule we aim to deliver in order to restore its depletion in macrophages, when associated with their death leading to plaque rupture in atherosclerotic lesions. Despite high interest in CS nanoparticles for drug delivery, due to the biodegradability of CS and to the ease of the preparation process, these systems tend to readily disintegrate when diluted in physiological media. Some stabilization strategies have been proposed so far but they typically involve the addition of a coating agent or chemical cross-linkers. In this study, we propose the complexation of CS with iron ions prior to nanoparticle formation as a strategy to improve the carrier stability. This can be achieved thanks to the ability of iron to strongly bind both chitosan and phosphate groups. Nanoparticles were obtained from either TPP or ATP and chitosan-iron (CS-Fe) complexes containing 3 to 12% w/w iron. Isothermal titration calorimetry showed that the binding affinity of TPP and ATP to CS-Fe increased with the iron content of CS-Fe complexes. The stability of these nanoparticles in physiological conditions was evaluated by turbidity and by fluorescence fluctuation in real time upon dilution by electrolytes, and revealed an important stabilization effect of CS-Fe compared to CS, increasing with the iron content. Furthermore, in vitro studies on two macrophage cell lines (J774A.1 and THP-1) revealed that ATP uptake is improved consistently with the iron content of CS-Fe/ATP nanoparticles, and correlated to their lower dissociation in biological medium, allowing interesting perspectives for the intracellular delivery of ATP.

Altered cerebral vascular volumes and solute transport at the blood-brain barriers of two transgenic mouse models of Alzheimer's disease.

We evaluated the integrity and function of the blood-brain barrier in 3xTg-AD mice aged 3-18 months and in APP/PS1 mice aged 8-months to determine the impacts of changes in amyloid and tau proteins on the brain vascular changes. The vascular volume (Vvasc) was sub-normal in 3xTg-AD mice aged from 6 to 18 months, but not in the APP/PS1 mice. The uptakes of [(3)H]-diazepam by the brains of 3xTg-AD, APP/PS1 and their age-matched control mice were similar at all the times studied, suggesting that the simple diffusion of small solutes is unchanged in transgenic animals. The uptake of d-glucose by the brains of 18-month old 3xTg-AD mice, but not by those of 8-month old APP/PS1 mice, was reduced compared to their age-matched controls. Accordingly, the amount of Glut-1 protein was 1.4 times lower in the brain capillaries of 18 month-old 3xTg-AD mice than in those of age-matched control mice. We conclude that the brain vascular volume is reduced early in 3xTg-AD mice, 6 months before the appearance of pathological lesions, and that this reduction persists until they are at least 18 months old. The absence of alterations in the BBB of APP/PS1 mice suggests that hyperphosphorylated tau proteins contribute to the vascular changes that occur in AD.

Towards an improved anti-HIV activity of NRTI via metal-organic frameworks nanoparticles.

Nanoscale mesoporous iron carboxylates metal-organic frameworks (nanoMOFs) have recently emerged as promising platforms for drug delivery, showing biodegradability, biocompatibility and important loading capability of challenging highly water-soluble drugs such as azidothymidine tryphosphate (AZT-TP). In this study, nanoMOFs made of iron trimesate (MIL-100) were able to act as efficient molecular sponges, quickly adsorbing up to 24 wt% AZT-TP with entrapment efficiencies close to 100%, without perturbation of the supramolecular crystalline organization. These data are in agreement with molecular modelling predictions, indicating maximal loadings of 33 wt% and preferential location of the drug in the large cages. Spectrophotometry, isothermal titration calorimetry, and solid state NMR investigations enable to gain insight on the mechanism of interaction of AZT and AZT-TP with the nanoMOFs, pointing out the crucial role of phosphates strongly coordinating with the unsaturated iron(III) sites. Finally, contrarily to the free AZT-TP, the loaded nanoparticles efficiently penetrate and release their cargo of active triphosphorylated AZT inside major HIV target cells, efficiently protecting against HIV infection.

Oatp1a4 and an L-thyroxine-sensitive transporter mediate the mouse blood-brain barrier transport of amyloid-ß peptide.

The influx of amyloid-ß peptide (Aß) across the blood-brain barrier is partly mediated by the receptor for advanced glycation end products (RAGE). But other transporters, like Oatp (organic anion transporter polypeptide, SLC21) transporters, could also be involved. We used in situ brain perfusion to show that rosuvastatin and taurocholate, two established Oatp1a4 substrates, decreased (5-fold) the Clup of [3H]Aß while L-thyroxine increased it (5.5-fold). We demonstrated an interaction between Aß and Oatp1a4 by co-immunoprecipitation and western blotting experiments, supporting the hypothesis that the rosuvastatin- and taurocholate-sensitive transporter was Oatp1a4. In conclusion, our results suggest that, in mice, the brain uptake of Aß is partly mediated by Oatp1a4 and that L-thyroxine may play a crucial role in the inhibition of brain Aß clearance.

Intestinal permeation enhancement of docetaxel encapsulated into methyl-ß-cyclodextrin/poly(isobutylcyanoacrylate) nanoparticles coated with thiolated chitosan.

In this study we investigated the potential of mucoadhesive nanoparticles to enhance the intestinal permeability of docetaxel (Dtx). These nanoparticles were composed of methyl-ß-cyclodextrin (Me-ß-CD) combined with poly(isobutylcyanoacrylate) and coated with thiolated chitosan. In order to encapsulate the highest amount of Dtx into nanoparticles, the anionic emulsion polymerization of isobutylcyanoacrylate was carried out in a solution of Me-ß-CD/Dtx inclusion complex. The resulting nanoparticles were spherical with diameters ranging from 200 to 400 nm, and positively charged. Depending on the formulation, the encapsulation efficiency of Dtx was 70-80%. In vitro experiments in simulated intestinal medium containing 1% w/v of pancreatin showed that Dtx was gradually released to reach 60% after 24h and 100% after 48 h. The capacity of these nanoparticles to enhance the flux of Dtx across the intestinal membrane was then investigated using the Ussing chamber technique. The intestinal permeation of Dtx loaded into nanoparticles was found to be higher than the ethanol control solution of Dtx. Interestingly, when mucoadhesive interactions between nanoparticles and the mucosa were avoided, the intestinal permeation of Dtx significantly decreased, confirming that the mucoadhesion of the nanoparticles was a mandatory condition to enhance the intestinal permeation of Dtx.

ABCG2- and ABCG4-mediated efflux of amyloid-ß peptide 1-40 at the mouse blood-brain barrier.

The accumulation of amyloid-ß peptide (Aß) in the brain is a critical hallmark of Alzheimer's disease. This high cerebral Aß concentration may be partly caused by impaired clearance of Aß across the blood-brain barrier (BBB). The low-density lipoprotein receptor-related protein-1 (LRP-1) and the ATP-binding cassette (ABC) protein ABCB1 (P-glycoprotein) are involved in the efflux of Aß across the BBB. We hypothesized that other ABC proteins, such as members of the G subfamily, are also involved in the BBB clearance of Aß. We therefore investigated the roles of ABCG2 (BCRP) and ABCG4 in the efflux of [3H] Aß1-40 from HEK293 cells stably transfected with human ABCG2 or mouse abcg4. We showed that ABCG2 and Abcg4 mediate the cellular efflux of [3H] Aß1-40. In addition, probucol fully inhibited the efflux of [3H] Aß1-40 from HEK293-abcg4 cells. Using the in situ brain perfusion technique, we showed that GF120918 (dual inhibitor of Abcb1 and Abcg2) strongly enhanced the uptake (Clup, µl/g/s) of [3H] Aß1-40 by the brains of Abcb1-deficient mice, but not by the brains of Abcb1/Abcg2-deficient mice, suggesting that Abcg2 is involved in the transport of Aß at the mouse BBB. Perfusing the brains of Abcb1/Abcg2- and Abca1-deficient mice with [3H] Aß1-40 plus probucol significantly increased the Clup of Aß. This suggests that a probucol-sensitive transporter that is different from Abca1, Abcb1, and Abcg2 is involved in the brain efflux of Aß. We suggest that this probucol-sensitive transporter is Abcg4. We conclude that Abcg4 acts in concert with Abcg2 to efflux Aß from the brain across the BBB.

Direct evidence of abca1-mediated efflux of cholesterol at the mouse blood-brain barrier.

We investigated the expression and function of Abca1 in wild-type C57BL/6, abca1(+/+), and abca1(-/-) mice brain capillaries forming the blood-brain barrier (BBB). We first demonstrated by quantitative RT-PCR and Western immunoblot that Abca1 was expressed and enriched in the wild-type mouse brain capillaries. In abca1(-/-) mice, we reported that the lack of Abca1 resulted in an 1.6-fold increase of the Abcg4 expression level compared to abca1(+/+) mice. Next, using the in situ brain perfusion technique, we showed that the [(3)H]cholesterol brain uptake clearance (Cl(up), µl/s/g brain), was significantly increased (107%) in abca1(-/-) mice compared to abca1(+/+) mice, meaning that the deficiency of Abca1 conducted to a significant decrease of the cholesterol efflux at the BBB level. In addition, the co-perfusion of probucol (Abca1 inhibitor) with [(3)H]cholesterol resulted in an increase of [(3)H]cholesterol Cl(up) (115%) in abca1(+/+) but not in abca1(-/-) mice, meaning that probucol inhibited selectively the efflux function of Abca1. In conclusion, our results demonstrated that Abca1 was expressed in the mouse brain capillaries and that Abca1 functions as an efflux transporter through the mouse BBB.

A new nanomedicine based on didanosine glycerolipidic prodrug enhances the long term accumulation of drug in a HIV sanctuary.

New nanomedicines could improve drug accumulation in HIV sanctuaries and ameliorate their antiretroviral efficiency. In this view, we propose herein a combined strategy based on a biomimetic prodrug of ddI and its formulation in well-characterized lipid nanoobjects. The glycerolipidic prodrug of ddI (ProddINP) has been synthesized and its bulk structure was characterized. An appropriate formulation of this prodrug has been designed using a rational approach combining different physicochemical techniques. The high incorporation ratio of the prodrug into dipalmitoylphosphatidylcholine (DPPC) bilayers was determined by DSC. Then two liposome preparation methods were compared, with respect to size, incorporation yield and molecular/supramolecular organization of vesicles. The best liposomal formulation of ProddINP has been checked to keep intact the anti-HIV activity of ddI. This formulation was finally compared to ddI after oral route in rat. The animal experiments evidenced the increase of ddI blood half life (3-fold) and its enhanced accumulation as prodrug form at 24h in numerous organs and especially intestine after administration of ProddINP in comparison with free drug. Finally, the tested liposomal formulation of ProddINP seems to be a promising approach to eradicate HIV infection from intestinal sanctuaries where the virus can concentrate.

Role of two efflux proteins, ABCB1 and ABCG2 in blood-brain barrier transport of bromocriptine in a murine model of MPTP-induced dopaminergic degeneration.

PURPOSE: MPTP-induced dopaminergic degeneration is an experimental model commonly used to explore Parkinson's disease. Cerebral drug transport by ABC transporters in MPTP models has never been reported. METHODS: We have investigated the transport of bromocriptine through the blood-brain barrier (BBB) in a MPTP model to understand the influence of the dopaminergic degeneration on ABCB1 and ABCG2. RESULTS: We have shown that in MPTP treated mice, bromocriptine is widely distributed to brain (2.3-fold versus control, p less than 0.001) suggesting either disruption of BBB or alteration of active efflux of the drug. In situ brain perfusion of [14C]- sucrose and [3H]-inulin did not evidenced a BBB disruption. Studies of ABCB1 and ABCG2 activity showed that MPTP intoxication did not alter their functionality. Conversely, ABCG2 expression studied on brain capillaries from MPTP-treated mice was decreased (1.3-fold, p less than 0.05) and ABCB1 expression increased (1.43-fold, p less than 0.05) as an off-setting of brain transport. CONCLUSIONS: These data demonstrate that MPTP intoxication does not alter the BBB permeability. However, bromocriptine brain distribution is increased in MPTP animals. Hence, MPTP may interact with another transport mechanism such as uptake and/or other efflux transporters. Inflammation and Parkinson's-like lesions induced by MPTP intoxication could lead to modification of drug pharmacokinetics and have clinical consequences, such as neurotoxicity.

Interactions between riluzole and ABCG2/BCRP transporter.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative fatal disease. Drugs used in this disease need to cross the blood-brain barrier (BBB). Only riluzole is approved for ALS treatment. We have investigated riluzole as a breast cancer resistance protein (BCRP) substrate by studying its brain transport in CF1 mdr1a (-/-) mice and its intracellular uptake on BeWo cells (human placental choriocarcinoma cell line). We have also investigated the effect of riluzole on BCRP expression level and on its activity using the prazocin as a test probe for brain transport and intracellular uptake. Assays on mdr1a (-/-) mice and BeWo cells showed a higher uptake of riluzole when pretreated with a BCRP inhibitor. After repeated doses of riluzole, BCRP activity was increased in CF1 mdr1a (-/-) mice, riluzole uptake was decrease and both BCRP expression and activity were increased in BeWo cells. In conclusion, we report in this study that riluzole is transported by BCRP at the BBB level and can enhance its function. These results taken with our previous studies on riluzole and P-glycoprotein show that drug-drug interactions between riluzole and efflux transporters substrates may occur at the BBB level and should be taken into account in future clinical trial design in ALS.

Interactions between antiparkinsonian drugs and ABCB1/P-glycoprotein at the blood-brain barrier in a rat brain endothelial cell model.

Parkinson's disease is a neurodegenerative disorder that requires treatment by dopaminergic agonists, which may be responsible for central side effects. We hypothesized that the efflux transporter ABCB1/P-glycoprotein played a role in brain disposition of antiparkinsonian drugs and could control central toxicity. We aimed to evaluate antiparkinsonian drugs as ABCB1 substrates and/or inhibitors in rat brain endothelial cells GPNT, in order to predict potential clinical drug-drug interactions. Among the antiparkinsonian drugs tested, levodopa, bromocriptine, pergolide and pramipexole were ABCB1 substrates. However, only bromocriptine could inhibit ABCB1 functionality with an IC(50) of 6.71 microM on Rhodamine 123 uptake and an IC(50) of 1.71 microM on digoxine uptake. Thus, bromocriptine at 100 microM is responsible for an increase of levodopa intracellular transport of about 2.05-fold versus control. Therefore, we can conclude that bromocriptine is a potent drug for medicinal interactions in vitro. Hence, in patients with Parkinson's disease, these results may be considered to optimise treatments individually.

Squalenoylation favorably modifies the in vivo pharmacokinetics and biodistribution of gemcitabine in mice.

Gemcitabine (2',2'-difluorodeoxyribofuranosylcytosine; dFdC) is an anticancer nucleoside analog active against wide variety of solid tumors. However, this compound is rapidly inactivated by enzymatic deamination and can also induce drug resistance. To overcome the above drawbacks, we recently designed a new squalenoyl nanomedicine of dFdC [4-N-trisnorsqualenoyl-gemcitabine (SQdFdC)] by covalently coupling gemcitabine with the 1,1',2-trisnorsqualenic acid; the resultant nanomedicine displayed impressively greater anticancer activity compared with the parent drug in an experimental murine model. In the present study, we report that SQdFdC nanoassemblies triggered controlled and prolonged release of dFdC and displayed considerably greater t(1/2) (approximately 3.9-fold), mean residence time (approximately 7.5-fold) compared with the dFdC administered as a free drug in mice. It was also observed that the linkage of gemcitabine to the 1,1',2-trisnorsqualenic acid noticeably delayed the metabolism of dFdC into its inactive difluorodeoxyuridine (dFdU) metabolite, compared with dFdC. Additionally, the elimination of SQdFdC nanoassemblies was considerably lower compared with free dFdC, as indicated by lower radioactivity found in urine and kidneys, in accordance with the plasmatic concentrations of dFdU. SQdFdC nanoassemblies also underwent considerably higher distribution to the organs of the reticuloendothelial system, such as spleen and liver (p < 0.05), both after single- or multiple-dose administration schedule. Herein, this paper brings comprehensive pharmacokinetic and biodistribution insights that may explain the previously observed greater efficacy of SQdFdC nanoassemblies against experimental leukemia.

Analysis of plasma protein adsorption onto PEGylated nanoparticles by complementary methods: 2-DE, CE and Protein Lab-on-chip system.

The biodistribution of colloidal carriers after their administration in vivo depends on the adsorption of some plasma proteins and apolipoproteins on their surface. Poly(methoxypolyethyleneglycol cyanoacrylate-co-hexadecylcyanoacrylate) (PEG-PHDCA) nanoparticles have demonstrated their capacity to cross the blood-brain barrier (BBB) by a mechanism of endocytosis. In order to clarify this mechanism at the molecular level, proteins and especially apolipoproteins adsorbed at the surface of PEG-PHDCA nanoparticles were analyzed by complementary methods such as CE and Protein Lab-on-chip in comparison with 2-D PAGE as a method of reference. Thus, the ability of those methodologies to identify and quantify human and rat plasma protein adsorption onto PEG-PHDCA nanoparticles and conventional PHDCA nanoparticles was evaluated. The lower adsorption of proteins onto PEG-PHDCA nanoparticles comparatively to PHDCA nanoparticles was evidenced by 2-D PAGE and Protein Lab-on-chip methods. CE allowed the quantification of adsorbed proteins without the requirement of a desorption procedure but failed, in this context, to analyze complex mixtures of proteins. The Protein Lab-on-chip method appeared to be very useful to follow the kinetic of protein adsorption from serum onto nanoparticles; it was complementary to 2-D PAGE which allowed the identification (with a relative quantification) of the adsorbed proteins. The overall results suggest the implication of the apolipoprotein E in the mechanism of passage of PEG-PHDCA nanoparticles through the BBB.