Chemically diverse nanoparticles form the basis of a number of emerging applications in engineering, including drug and gene delivery vehicles. However, it is difficult to predict the optimal chemical and physical properties for delivery of a specific drug or biomolecule. A key challenge in nanoparticle development is the need to perform distinct synthesis, characterization, and formulation steps before performance can be evaluated. Combinatorial, high-throughput (HTP) methods can aid in the discovery of new nanoparticles for delivery, while simultaneously elucidating important design parameters of a given system for a targeted application.
We will present a "modular" approach that applied combinatorial Controlled Radical Polymerization (CRP)1-2 and ring-opening polymerization (ROP) for the formation, characterization, and screening of more than 1,800 core cross-linked nanoparticles for siRNA delivery.3 Combinatorial methods have driven small molecule drug discovery,4 but have been less explored for the discovery of polymers5 for specific applications in part due to the inherent challenge in synthesizing and characterizing such complex materials. Advances made in the controlled synthesis of polymers (particularly CRP and functional ROP methods) and in robotic technology motivated us to examine physical and chemical properties in relation to performance.
We aimed to prepare a library of structurally distinct nanoparticles comprised of cationic cross-linked cores and variable shells with precise control over the particle size, chemical composition, and architecture. siRNA can complex to or inside of the core, while the hairy arms provide a protective shield. To achieve the desired structure, we employed a method of cross-linking block copolymers prepared by Reversible Addition-Fragmentation chain Transfer (RAFT) polymerization and ROP of functional polyesters, building on previously published reports.6-10 For the cross-linking, we sought an efficient and versatile reaction that would be permit library formation. We chose the ring-opening reaction of amines with epoxides9,10 due to the availability of a large number of amines that can incorporate cationic charge into the core, and the efficiency and high rate of catalyst-free reaction.
Analysis revealed structure-function relationships and beneficial design guidelines, including a higher reactive block weight fraction, stoichiometric equivalence between epoxides and amines, and short hydrophilic shells. Cross-linkers optimally possess tertiary dimethylamine or piperazine groups, and potential buffering capacity. Covalent cholesterol attachment allowed for transfection in vivo to liver hepatocytes and blood immune cells.
To examine the effect of block length on nanoparticle formation, a series of block copolymers were synthesized by RAFT that varied in weight % of oligoethylene oxide methacrylate (OEOMA) and glycidyl methacrylate (GMA) blocks. After cross-linking with different amines, optimal particle size (10-100 nm) was obtained for most block copolymers, except for when the GMA block was less than 5 wt.% or greater than 70 wt.%. In addition, ROP of allylglycolide from monomethoxy PEG initiators was conducted to prepare biodegradable nanoparticles. Oxidation to epoxides was performed using m-chloroperoxybenzoic acid (mCPBA).
We found that the block length, concentration, temperature, and choice of cross-linker all play roles in the formation of the hairy nanoparticles. AFM images of nanoparticles formed from the cross-linking of P(GMA39‐b‐(OEO5MA)41) with the linear aliphatic 1,10-diaminodecane show uniform particles with a diameter of 34 nm. Polyamines were also able to efficiently cross-link the block copolymers to form the hairy nanoparticle shape. The reaction of P((OEO5MA)25-b‐GMA10) with pentaethylenehexamine formed particles with a diameter of 21 nm as measured by DLS and 25.4 nm by AFM. To verify the core-shell structure, transmission TEM was employed. An examination of these particles revealed a higher electron density in the core, compared with the shell, indicating the core cross-linked morphology. Culturing fluorescently labeled particles with HeLa cells showed that they could be efficiently internalized.
After completion of preliminary studies, the synthesis were scaled up by reacting 19 different blocks copolymers with more than 100 amines in a combinatorial manner on a Symyx fluid handling robot to yield >1,800 structurally distinct core-shell nanoparticles. We characterized these nanoparticles using HTP GPC. We then screened them for siRNA complexation, siRNA delivery, and pDNA delivery in vitro. Many nanoparticles were able to complex tightly to siRNA and effectively deliver siRNA in vitro, silencing more than 80% of luciferase expression. The best performing nanoparticles were tested in vivo and were capable of silencing more than 50% of Factor VII in mice. In a separate experiments, these siRNA-loaded nanoparticles silenced >30% of CD45 protein expression in the blood monocyte/macrophage (CD11b+ cells) population.
We believe that the development of this library of core-shell nanoparticles represents an important step forward for the HTP synthesis of polymers. We aimed to move beyond simple linear polymers by exploring CRP and ROP for the formation of complex architectures with great chemical diversity in the core and shell. This one pot synthetic method enables parallel generation of large libraries of chemically distinct core-shell materials. Notably, a number of materials were identified with both in vitro and in vivo utility and the common structural features of these materials suggest certain design criteria for creating future intracellular delivery agents. These trends, full synthetic details and characterization, and in vivo biodistribution and knockdown data will be presented.
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