Nanoparticle shell structural cues drive in vitro transport properties, tissue distribution and brain accessibility in zebrafish
Graphical abstract
Introduction
Poly(ethylene glycol) (PEG) is known to provide colloidal stability and to tune the pharmacokinetic properties of drug nanocarriers [1]. PEG chains grafted at the surface of nanoparticles (NPs) enhance their steric stabilization and drastically reduce the rate and the extent of non-specific protein adsorption on their surface. However, PEG presents some shortcomings as it does not prevent completely protein adsorption [2]. Moreover, recent reports have shown that PEG can induce immune and inflammatory reactions in patients, inducing unwanted adverse effects and accelerated blood clearance of nanomedicine [3,4].
Alternatives to PEG in nanomedicine formulations have been thus actively investigated, and many candidates have been suggested, including zwitterionic polymers [5,6]. Amongst the proposed zwitterionic polymers, Poly(methacryloyloxyethyl phosphorylcholine) (PMPC) stands out for its unique biomimetic and antifouling properties. PMPC is constituted of a methacrylate backbone bearing phosphorylcholine side groups. The phosphorylcholine group is a zwitterion with the presence of anionic phosphate ions and a cationic tertiary amine group. PMPC possesses distinctive advantages: 1) biomimetism, the phosphorylcholine group being one of the most abundant head-group amongst cell membrane phospholipids; 2) the hydration state of the zwitterionic group providing excellent antifouling properties, and 3) the chemical stability of the methacrylate backbones. Because of his unique hydration, PMPC does not interact strongly with proteins or cells [7,8]. Zwitterionic polymer coatings have been shown to generate the best performance in alleviating the immune system activation compared to PEG [[9], [10], [11]]. PMPC and copolymers containing MPC have been developed initially for biomedical applications such as coatings to reduce protein adsorption, to reduce platelet adhesion and activation on cardiovascular devices, or to provide lubrication to orthopedic devices such as hip joint [12,13].
PMPC has also been successfully used to prepare NPs. Polystyrene nano-spheres surface-modified with PMPC were described as early as two decades ago by Uchida et al. [14]. More recently, poly(2-methacryloyloxyethyl phosphorylcholine)-b-poly(2-(diisopropylamino)ethyl methacrylate) (MPC-DPA) nanoparticles encapsulating curcumin were proposed by Salvage et al. [15]. The preparation of core-shell NPs has also been proposed based on diblock polyester polymer, a hydrophobic polyester block to form the biodegradable, hydrophobic matrix core for drug encapsulation, and a hydrophilic PMPC block to form the particle shell. However, reports examining the benefits of PMPC coating on NPs biological fate are scarce. Phosphorylcholine-derived zwitterion polymer as particle coating material showed strong anti-fouling properties [[16], [17], [18]]. Protein adsorption on PMPC-modified flat surface showed dependence over chain grafting density and chain length with a stronger dependence on the former than the latter [17]. Wang et al. recently showed that PMPC provides to albumin NPs an extended circulation time and improved tumor penetration [19]. On the other hand, Lalloz et al. observed that NPs skin penetration was not affected by the nature of the hydrophilic coating, as similar penetration data were obtained for PEG-coated and PMPC-coated NPs [20].
Improved drug delivery to the brain is a long-identified need. This need is particularly acute for long-term neurodegenerative diseases (NDD) and dementia such as Alzheimer's disease (AD) which could require a chronic treatment and repeated administrations. Delivery mediated by nanosized vehicles has been proposed as a solution to overcome the shortcomings of current drugs by reducing the number of administrations while increasing their efficacy [21,22]. However, NPs administrated intravenously encounter multiple physiological barriers including, protein opsonization, macrophage uptake, and the blood-brain barrier (BBB), and are unlikely to reach the brain without be carefully designed (Fig. 1). So far, the capacity of PMPC-coated NPs to cross biological barriers has not been reported and deserves special attention. Furthermore, it is not clear at this time if PMPC-coated NPs could exhibit similar biodistribution compared to PEG-coated NPs and could therefore be used as a promising alternative to PEG.
Zwitterion polymers stand out as the first choice for PEG nanocarrier coating [5,23]. Zwitterion polymers possess highly hydrated monomer with exceptional antifouling properties. However, until now, very few direct comparisons have been published, comparing neutral uncharged PEG coating with neutral charged zwitterion polymer [19,20,24,25]. This direct comparison, using same methodologies is critical to get fair appraisal of both type of coating. Moreover, the issue of body biodistribution as well as cell barrier crossing of zwitterion-coated nanocarriers have yet to be addressed. Finally, we introduced for the first time, a new technique, namely nanoscale flow cytometry, to study and quantify particles translocation as well as particle/protein interactions.”
The objectives of the study were to unravel, for the first time, the similarity and difference of PEG and PMPC coating for nanoparticles behavior in vitro and in vivo. The goal being to determine whether brain targeting of nanocarriers could benefit from zwitterionic coating. In this work, NPs either prepared from PMPC-b-PLA or PEG-b-PLA polymers were compared in a series of in vitro and in vivo assays aimed to provide mechanistic insights on how these NPs interact with proteins, cells, cell barriers as the blood-brain barrier as well with neuronal and macrophage cells (Fig. 1).
Section snippets
Materials
All the fine chemicals used for the polymers synthesis were from Sigma-Aldrich (ON Canada); organic solvents were from Fisher Scientific (Fisher Canada). Cell culture media and Fetal Bovine Serum (FBS) were from Wisent (Canada); bEnd.3 cells (murine vascular endothelial cell line), Raw 264.7 (murine macrophage), N2a (murine neuronal cell line) were from ATCC (ATCC, USA). N11 mouse microglia immortalized cell lines was a generous gift of Pr P. Talbot (INRS, Centre Armand-Frappier
Nanoparticles characterization
Synthesis and characterizations of the polymers used in this study are reported in Supporting Information (Table S1), including PEG-b-PLA [26], PMPC-b-PLA [20,36,37] and fluorescent Cy5-PLA.
Batches of NPs were prepared by "flash" nanoprecipitation (impinging jet micromixer) method to produce PEGylated and PMPC-coated NPs with similar hydrodynamic diameters in a robust manner [29]. Size control was achieved by adjusting injection flow rates and polymer concentration in the organic phases.
Shell engineering enables fine-tuning of NPs biodistribution
NPs capture by the endothelium has profound implications on NPs pharmacokinetics and biodistribution. The presented data on biodistribution of NPs in zebrafish larvae proved to some extend that independently of their shell composition, NPs preferentially accumulate in regions of low blood flow rate, especially in tortuous capillaries where flow rates are at least an order of magnitude smaller than those found in straight capillaries such as arterioles [66]. Besides this general observation, the
Conclusion
This comprehensive study illustrates the underestimated role of neutral surface chemistry (neutral uncharged vs. neutral charged surface) on the nano-bio-interface and their consequences on biodistribution in vivo. Besides their similar size and surface potential, side-by-side comparison of PEG- and PMPC-coated NPs demonstrated for the first time their remarkable differences in vitro and in vivo. Unexpectedly, PMPC coating was shown to increase protein binding, thank to advances in nanoscale
Data availability
The raw/processed data required to reproduce these findings cannot be shared at this time due to technical or time limitations.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
JMR thanks NSERC/CRSNG (Government of Canada) for postdoctoral fellowship (2017-2019). This research was undertaken thanks, in part, to funding from the Canada First Research Excellence Fund through the TransMedTech Institute (JMR postdoctoral fellowship, 2020-2021). CR acknowledges funding from NSERC/CRSNG (Government of Canada). XB acknowledges the support from the Canada Research Chair program and NSERC/CRSNG. JF is thankful for the financial support of the Arthritis Society (doctoral
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