Research Article
Pharmaceutical Nanotechnology
Spontaneous Self-Assembly of Polymeric Nanoparticles in Aqueous Media: New Insights From Microfluidics, In Situ Size Measurements, and Individual Particle Tracking

https://doi.org/10.1016/j.xphs.2016.09.024Get rights and content

Abstract

Supramolecular cyclodextrin-based nanoparticles (CD-NPs) mediated by host-guest interactions have gained increased popularity because of their “green” and simple preparation procedure, as well as their versatility in terms of inclusion of active molecules. Herein, we showed that original CD-NPs of around 100 nm are spontaneously formed in water, by mixing 2 aqueous solutions of (1) a CD polymer and (2) dextran grafted with benzophenone moieties. For the first time, CD-NPs were instantaneously produced in a microfluidic interaction chamber by mixing 2 aqueous solutions of neutral polymers, in the absence of organic solvents. Whatever the mixing conditions, CD-NPs with narrow size distributions were immediately formed upon contact of the 2 polymeric solutions. In situ size measurements showed that the CD-NPs were spontaneously formed. Nanoparticle tracking analysis was used to individually follow the CD-NPs in their Brownian motions, to gain insights on their size distribution, concentration, and stability on extreme dilution. Nanoparticle tracking analysis allowed to establish that despite their non-covalent nature, and the CD-NPs were remarkably stable in terms of concentration and size distribution, even on extreme dilution (concentrations as low as 100 ng/mL).

Introduction

Supramolecular nanostructures mediated by host-guest interactions based on cyclodextrins (CDs) have gained increased popularity for their potential biomedical applications, particularly in drug delivery.1, 2, 3 Drug loadings were dramatically improved when including CDs in the nanoparticles (NPs), due to complex formation between CDs and drugs.4 As an example, the loading of prednisolone in poly(alkyl cyanoacrylate) NPs was improved 129-fold when incorporated in CDs.5

More recently, CD-containing NPs (CD-NPs) were prepared by a mild, solvent-free method, at room temperature and without using surfactants.6 CD-NPs were spontaneously formed by mixing 2 aqueous solutions of (1) dextran (Dex) grafted with alkyl side chains (Dex-C12) and (2) a highly soluble CD polymer (poly-CD). The alkyl side chains of Dex-C12 formed inclusion complexes with the CDs in poly-CD, leaving most of the CDs free for the inclusion of other molecules of interest, such as anticancer drugs, contrast agents, nitric oxide donors, and cosmetic ingredients.1, 3 Based on a similar “lock and key” concept, the first small interfering RNA delivery system in humans using CDs was developed.7 The self-assembly of cationic poly-CDs with anionic nucleic acids gave rise to the formation of NPs to which poly(ethylene glycol) and targeting agents could be associated.7, 8

Benzophenone (Bz) was the most studied active ingredient in the NPs made of poly-CD and Dex-C12.6, 9, 10, 11, 12, 13, 14 Attractive features of the Bz-loaded NPs were their one step solvent-free preparation method, the small sizes and the possibilities to be freeze-dried and reconstituted in aqueous solutions.6, 14 Bz was incorporated in the polymeric solution and it was retained in the NPs as inclusion complexes with the CDs.13 However, because of the competition between Bz and the alkyl chains for the CD cavities, Bz loadings reached at best 2.9 wt% and there was a significant amount of free Bz in the NP suspensions.13

We propose here an original approach to prepare Bz-containing CD-NPs, avoiding the presence of free Bz in the suspensions. Bz was grafted to Dex, replacing the alkyl side chains. The interaction of this novel conjugate (Dex-Bz) with poly-CD, leading to CD-NP formation, was investigated by a set of complementary novel characterization techniques. For the first time, CD-NPs were instantaneously produced in a microfluidic interaction chamber by mixing 2 aqueous solutions of neutral polymers, in the absence of any organic solvent.

Interestingly, an in situ method was developed to study the CD-NP formation directly in the preparation vessel. Nanoparticle tracking analysis (NTA) was used to individually follow the NPs in their Brownian motion to gain insight into their size distribution, concentration, and stability upon extreme dilution.

Section snippets

Materials

Dex (40,000 g/mol) was purchased from Pharmacia (Uppsala, Sweden). β-CD was kindly provided by Roquette (Vecquemont, France). 1-Adamantyl chloride and 4-benzoylbenzoic acid were obtained from Sigma Aldrich (Saint-Quentin-Fallavier, France). MilliQ water was purified by reverse osmosis (Millipore®, Billerica, MA) and filtered using a 0.22-μm membrane (Millipore). All organic solvents were analytical grade and obtained from Thermo Fisher (Illkirch, France).

Synthesis of Modified Dextran Polymers

To synthesize Dex bearing Bz side units

Preparation of Poly-CD/Dex-Bz NPs by Microfluidic Device

Dex grafted with Bz moieties was successfully synthetized. After synthesis, Dex-Bz samples were extensively purified by dialysis and characterized by SEC and 1H NMR spectroscopy. The average molar mass was shifted from 37,700 ± 400 (native Dex) to 43,400 ± 4300 g/mol, after Bz grafting. The increase in molar mass was a clear indication of the successful grafting reaction. 1H NMR enabled to determine the substitution yield, showing that 5% of the Dex glucose units were effectively grafted with

Conclusion

NPs were spontaneously formed by mixing 2 aqueous polymer solutions. A microfluidic device has been set up to produce NPs of around 100 nm, with narrow size distributions. In situ size measurements showed that the NPs were spontaneously formed. NTA allowed to individually track each NP in its Brownian motion, showing that despite dilution up to 100 ng/mL, the NPs were still stable in terms of mean diameters. The NP number was proportional to the dilution factor, showing that the NPs did not

Acknowledgments

We acknowledge Gesine Heuck (Precision NanoSystems, Inc.) and Nicolas Gonzalez (Schaefer Technologies) for helpful discussions concerning the microfluidics experiments. We thank Sylvain Boj (Cordouan) for in situ analysis using VASCO-FlexTM system. We are grateful to Dr. Catherine Ladavière (IMP, Lyon) for her kind help with the characterization of Dex-Bz conjugates. We acknowledge support from China scholarship council (CSC, no. 201408330166), China National Science and Technology Major

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