Functionalization of Cellulose Nanocrystals with POEGMA Copolymers via Copper-Catalyzed Azide–Alkyne Cycloaddition for Potential Drug-Delivery Applications

Abstract

Elongated colloidal nanoparticles (NPs) have significant potential for drug delivery and imaging applications in cancer therapy, but progress depends on developing a deeper understanding of how their physicochemical properties affect their interactions with cells and with tumors. Cellulose nanocrystals (CNCs) are biocompatible, rodlike colloids that are broadly surface-functionalizable, making them interesting as modular drug carriers. In this report, we describe the attachment of a statistical copolymer containing oligoethylene glycol methacrylate (OEGMA; M n ≈ 500 Da) and small amounts of aminopropylmethacrylamide (APMA) to CNCs. Here, the copolymer is designed to serve as a "stealth" corona to minimize protein adsorption, and the amino groups provide functionality for the attachment of diagnostic or therapeutic moieties. The corona polymer with a terminal azide group was synthesized by atom transfer radical polymerization using tertbutyloxycarbonyl (tBoc)-protected APMA as the comonomer. A key step in this synthesis was the grafting of acetylene groups to the CNC surface via a reaction with NaOH plus propargyl bromide in aqueous dimethyl sulfoxide. The copolymer was attached to the CNCs using copper-catalyzed azide−alkyne cycloaddition (CuAAC) "click" chemistry. By determining the mean number of amino groups per copolymer and amino group content of the CNC sample, we were able to infer that there were on average ca. 300 polymer molecules per CNC. Preliminary evaluation in a human ovarian cancer cell line (HEYA8) and a human breast cancer cell line (MDA-MB-436) demonstrated that these CNCs are nontoxic. We also assessed the cellular uptake of these CNC NPs in the same two cell lines using flow cytometry and distinguished between NPs being internalized by the cell or surface-bound using a trypan blue quenching experiment. These results provide support for applications of polymer-coated CNCs in medicine and are encouraging for further studies in vitro and in vivo to evaluate their potential as drug-delivery vehicles.

Changing Surface Polyethylene Glycol Architecture Affects Elongated Nanoparticle Penetration into Multicellular Tumor Spheroids

“…The length data for this CNC sample are presented in ref . One sample of CNCs was modified to have alkyne groups (CNC–) on the surface, and the second sample was modified to have amine surface functionality (CNC–NH 2 ). The CNC– NPs were end-grafted with a POEGMA copolymer (N 3 -PAPMA 2 - stat -POEGMA 30 ) using a Cu­(I)-catalyzed the azide–alkyne coupling reaction.…”

“…The mean separation of anchor points for the POEGMA polymer on the PFS micelles should also be close to 3 nm. We measured the hydrodynamic size of FITC-labeled PAPMA 2 - stat -POEGMA 30 by DLS and found a z -average hydrodynamic radius R h = 4.7 nm. We estimated R h for the DOTA-containing PGlu 20 - b -PEG 48 by comparing its retention volume in a size exclusion chromatography measurement with those of a series of PEG standards of known R h and calculated a value of 3.2 nm …”

Solvent-Mediated Isolation of Polymer-Grafted Nanoparticles

“…Figure shows schematically the synthesis of polymer-grafted CNCs and their purification from the free (nonattached) polymer. Azide-terminated poly­( N -isopropyl acrylamide) (pNIPAm) was attached to alkyne-modified CNCs by the “grafting-to” method using a copper-catalyzed click reaction. , The pNIPAm molecules with the molecular weight of 19.3 and 66.1 kDa were synthesized by reversible addition-fragmentation chain-transfer (RAFT) polymerization using an azido-RAFT agent (2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid 3-azido-1-propanolester), which introduced an azido group to one end of the pNIPAm chains (Table S1). The synthesis of the polymer-grafted CNCs is described in Supporting information, Section S2.…”

“…Alkyne-functionalized CNCs were prepared using a previously reported method . Briefly, 0.288 g of NaOH was added to 250 mL of 6 wt % CNC suspension in 1:1 v/v dimethyl sulfoxide (DMSO)/water mixture.…”

“…Alkyne-functionalized CNCs were prepared using a previously reported method. 49 Briefly, 0.288 g of NaOH was added to 250 mL of 6 wt % CNC suspension in 1:1 v/v dimethyl sulfoxide (DMSO)/water mixture. Subsequently, 802 μL of propargyl bromide was added, and the suspension was stirred at room temperature for 48 h. Next, the suspension was heated to 50 °C and stirred for 48 h. The suspension was then dialyzed against Milli-Q grade distilled deionized water (DI, 18.2 MΩ cm resistivity) for 5 days, changing the water twice a day.…”