A mechanistic investigation on methotrexate-loaded chitosan-based hydrogel nanoparticles intended for CNS drug delivery: Trojan horse effect or not?
Graphical abstract
Introduction
Central nervous system (CNS) requires an extremely controlled environment to keep its unique homeostasis including nutrients, protein content, ions and immune balance [1]; hence, a sophisticated multi-layered mechanism has evolved to form the CNS barriers. In the first level, tight junctions sealing the endothelium of brain capillaries and epithelial cells of choroid plexus shape the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) respectively to restrict intercellular trafficking of cells, large molecules and hydrophilic substances. In the second level, settlement of metabolising enzymes and efflux transporters within the endothelium prevent the unwished small and large molecules and viruses that had passed the first level from reaching the nerve cells. Finally, pericytes and astrocytes end-feet build the third protective shield of the neurons [2].
Although these mechanisms evolved for optimum protection of CNS, in case of drug delivery they are undesirable. One of the most powerful components of this protective shield is P-glycoprotein (P-gp). These transporters are localized in luminal side of endothelial cells, said to be responsible for ATP-dependent efflux of an astonishing number of therapeutics different in size, structure and hydrophobic nature, including many groups of anticancer agents. Soon after they have been discovered, utilising a P-gp inhibitor became a routine strategy for experiments on brain drug delivery, at least in the laboratory [3,4].
The two past decades have witnessed a revolution in the art of drug delivery, immediately after nanotechnology potentials were considered in medical sciences. Hydrogel nanoparticles or nanogels are among the most promising polymeric nanoparticles because of their appealing characteristics including hydrophilic nature, a significant capacity of water absorption, flexibility, biocompatibility, long presence in blood circulation and the possibility of being exploited for passive and active targeting. Chitosan produced by deacetylation of chitin forms positively-charged hydrogel nanoparticles that have received enormous academic attention for drug delivery purposes because of negligible toxicity, muco-adhesiveness and their ability to open tight junctions, besides the typical advantages of hydrogel nanoparticles [5,6]. In this regard, our research group has previously characterised and optimised a chitosan nanogel formulation for brain delivery of the poor BBB-permeating methotrexate (MTX). It is reported that methotrexate, when carried in a chitosan based nanogel formulation, produces a significantly higher brain concentration both in intravenous and intranasal administration in comparison with administration of the commercial simple solution of methotrexate in the same route [[7], [8], [9], [10], [11]].
MTX, a folic acid analogue, is one of the most widely used antineoplastic agents. It is a key therapeutic in head and neck carcinomas and the first line medication in primary brain lymphomas because of remarkable increase in patients' survival; nevertheless, it is substrate of various efflux transporters including BCRP, MRP1-7, OAT1-4, OATP1B1, OATP1B3, OATP1C1 and especially P-gp and hence passes through BBB very poorly because of its tiny hydrophilic molecule. As MTX passage through BBB is dose-dependent, a high dose systemic injection or intrathecal administration is required in order to maintain the therapeutic dose in the brain; hence, significant neurotoxicity and nephrotoxicity is expected following an MTX-based cancer chemotherapy regimen [[12], [13], [14], [15], [16]].
It has been revealed that chitosan hydrogel nanoparticles provide a noticeably higher concentration of MTX in the brain in comparison to its commercial simple solution when administered intravenously [9]. A possible hypothesis is that chitosan nanoparticles release their MTX content in the vicinity of BBB leading to higher BBB passage, P-gp transporters saturation and an ultimate higher brain accumulation. Another plausible proposition is that chitosan nanoparticles pass through BBB themselves and release their MTX just in the nerves purlieus. To figure out what occurs in vivo, MTX commercial solution and MTX containing chitosan nanoparticles administered intravenously for rats after a verapamil dose, that is a P-gp competitive inhibitor [17,18].
Section snippets
Materials
Acetonitrile HPLC grade (Samchun Chemicals, Korea), low molecular weight chitosan (Primex, Island), MTX solution for injection 100 mg/mL (Mylan, France), tripolyphosphate (TPP; Merck, Germany), and trichloroacetic acid (TCA; Merck, Germany) were utilised through this study. Besides, male Sprague-Dawley rats weighing 200–250 g were subject to the present animal experiments.
MTX quantification
In the present study, MTX was quantified in aqua, plasma and brain environments with the validated reverse phased
MTX quantification
MTX is a small hydrophilic molecule that passes through BBB very poorly with a dose-dependent mechanism; hence a validated precise analytical method is provided for its quantification, where used in vivo and in small doses. In this regard, a previously developed HPLC method for MTX analysis in the aqueous environment, brain and plasma in the range of 25–1000 ng/mL was exploited [19].
Nanogel preparation and characterisation
Ionic gelation method takes advantage of the opposite electrical charges of a gel-forming polymer like chitosan,
Conclusions
Chitosan-based hydrogel nanoparticles with a mean diameter of <200 nm have been widely investigated for CNS drug delivery purposes especially in case of poor BBB passing drugs and via various routes such as intravenous and intranasal administration. It has been discovered that these nanogels provide a significantly higher concentration of the drug in the brain in comparison with the free drug solution. Even though, it has not been apparent what happens from a pharmacokinetic view to make the
Conflicts of interest
This study was funded by Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences (grant number: 11725-103-01-95).
The authors report no conflict of interest.
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