NOVEL METHOD OF POLYMERIC NANOPARTICLE FABRICATION FOR CANCER TREATMENT AND OTHER DRUG DELIVERY APPLICATIONS

Abstract

A novel and innovative method of fabricating nanoparticles with reproducible characteristics from batch-to-batch and during scale-up. The method is a dipolymerization-precipitation reaction facilitated by the inverse electron demand Diels-Alder (IEDDA) reaction.

Claims

1. A method of fabricating nanoparticles with reproducible characteristics, comprising a dipolymerization-precipitation reaction facilitated by the inverse electron demand Diels-Alder (IEDDA) reaction.

2. A method of fabricating nanoparticles with reproducible characteristics, comprising functionalizing HO-PLGA-PEG (HO-poly (lactide-co-glycolide)-polyethylene glycol) or HO-PLGA (HO-poly (lactide-co-glycolide) with trans-cyclooctene (TCO) to yield TCO-PLGA-PEG or TCO-PLGA, respectively, and dissolving TCO-PLGA-PEG or TCO-PLGA in a solvent mixture to produce a polymer dimer with double the molecular weight and thus insoluble in the solvent mixture.

3. A method of fabricating nanoparticles with reproducible characteristics, comprising functionalizing HO-PLGA (HO-poly (lactide-co-glycolide) with tetrazine to yield Tetrazine-PLGA, and dissolving Tetrazine-PLGA in a solvent mixture to produce a polymer dimer with double the molecular weight and thus insoluble in the solvent mixture.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0009] The present method is a novel method to prepare nanoparticles to facilitate excellent batch-to-batch reproducibility during nanoparticle fabrication and scale-up using the inverse electron demand Diels-Alder (IEDDA) reaction to facilitate dipolymerization followed by nanoparticle precipitation in a mixture of solvents. Using this strategy, it is the solvent composition among other formulation variables at the point of polymer coupling that determines polymer precipitation and particle formation. In the present method, this variable can easily be controlled from batch to batch and with scale-up. Optimization of the nanoparticle formation process by varying formulation and process variables will lead to reproducible nanoparticle characteristics.

[0010] The present inventor has contributed to the use of dispersion polymerization for drug delivery applications (Adesina et al., “Optimization of the fabrication of novel stealth PLA-based nanoparticles by dispersion polymerization using D-optimal mixture design”, Drug Development and Industrial Pharmacy, 2013, 40(11): 1547-1556; Adesina et al., “Polylactide-based Paclitaxel-loaded Nanoparticles Fabricated by Dispersion Polymerization: Characterization, Evaluation in Cancer Cell Lines, and Preliminary Biodistribution Studies”, Journal of Pharmaceutical Sciences, 2014, 103(8): 2546-2555; Akala and Adesina, “Chapter 1—“Fabrication of polymeric core-shell nanostructures” Nanoscale Fabrication, Optimization, Scale-Up and Biological Aspects of Pharmaceutical Nanotechnology”, Pages 1-49, Edited by Alexandru Mihai Grumezescu. Elsevier Inc. (2018) ISBN: 978-0-12-813629-4; the content of all of which is incorporated herein by reference). In dispersion, the starting reaction mixture is a clear, single-phase solution; particles are formed by precipitation of growing polymer chains. Therefore, the solvent medium becomes a dispersion medium (Horak, “Effect of reaction parameters on the particle size in the dispersion polymerization of 2-Hydroxyethyl Methacrylate”, J. Polym. Sci. A Polym. Chem., 1999, 37: 3785-3792; Capek, “Surface active properties of polyoxyethylene macromonomers and their role in radical polymerization in disperse systems”, Adv. Colloid Interface Sci., 2000, 88(3): 295-357; Leobandung et al., “Monodisperse Nanoparticles of Poly(ethylene glycol) Macromers and N-Isopropyl Acrylamide for Biomedical Applications”, J. Appl. Polym. Sci., 2003, 87:1678-1684; Song et al., “Monodisperse, controlled micron-size dye-labeled polystyrene particles by two stage dispersion polymerization”, Polymer, 2006, 47: 817-825; Ha et al., “Size Control of Highly Monodisperse Polystyrene Particles by Modified Dispersion Polymerization”, Macromol. Res., 2010, 18(10): 935-943).

[0011] Nanoprecipitation requires the addition of two solvents that are miscible with each other and the method results in instantaneous formation of nanoparticles, is easy to perform, can be easily scaled up and is a one-step procedure (Yadav et al., “Modified Nanoprecipitation Method for Preparation of Cytarabine-Loaded PLGA Nanoparticles”, AAPS PharmSciTech., 2010, 11(3): 1456-1465; the content of which is incorporated herein by reference). In nanoprecipitation, polymer and drug are dissolved in a water miscible organic solvent, for example, acetone or methanol. The solution is then added into an aqueous solution which contains a surfactant in a drop-wise manner. Through rapid solvent diffusion, the nanoparticles are formed immediately. The solvent is then removed under reduced pressure (Wang et al., “Manufacturing Techniques and Surface Engineering of Polymer Based Nanoparticles for Targeted Drug Delivery to Cancer”, Nanomaterials, 2016, 6, 26; the content of which is incorporated herein by reference). The size uniformity of polymeric nanoparticles is the most significant parameter that decides the consistency of performance.

[0012] Nanoprecipitation yields particles with broad particle size distribution affecting the consistency of performance which affects clinical translation (Chidambaram et al., “Modifications to the Conventional Nanoprecipitation Technique: An Approach to Fabricate Narrow Sized Polymeric Nanoparticles”, Adv. Pharm Bull., 2014, 4(2): 205-208). Particles with broad particle size distribution leads to difficulty in establishing the size of particles responsible for the biological effects. With dispersion polymerization on the other hand, although the reaction belongs to solution polymerization before the stage of the nucleation, the polymerization and polymer particle growth occur within the particles and, consequently, it is difficult to obtain uniform particles and uniform size distribution. In addition, the requirement of low monomer concentration and/or special processes hinder the practical application of precipitation polymerizations and these requirements are essential to avoid the coagulation of the resultant polymer particles (Liu et al., “Self-Stabilized Precipitation Polymerization and Its Application”, Research, 2018, Volume 2018, Article ID 9370490, 12 pages). Chemical initiators used in dispersion polymerization may also confer toxicity.

[0013] To ensure particle size uniformity, reproducibility and facilitate clinical use of polymeric nanoparticle platforms, Inverse Electron Demand Diels-Alder (IEDDA) chemistry is used to facilitate dipolymerization followed by precipitation. The IEDDA reaction is used between dienes, e.g. tetrazines, and strained dienophiles (e.g. trans-cyclooctene, TCO) in a bioorthogonal reaction (Oliveira et al., “Inverse Electron Demand Diels-Alder Reactions in Chemical Biology”, Chem. Soc. Rev., 2017, 46, 4895-4950; the content of which is incorporated herein by reference). Other bioorthogonal reactions such as strain promoted alkyne-azide cycloaddition or other forms of click chemistry or any other polymer coupling approach using various polymer coupling chemistries process may be used to prepare nanoparticles). Thus, the “Adesina method” is a dipolymerization-precipitation method or polymer coupling method of nanoparticle preparation described herein. The Adesina method is the method of the invention. The [4+2] cycloaddition of 1,2,4,5-tetrazines and various dienophiles such as TCO is very fast (10,000 times faster than the copper-catalyzed click reaction), selective, biocompatible and catalyst-free (Oliveira et al., supra). It is safe and biocompatible that it can be used in vivo for radiolabeling using pretargeting methodologies. Because of its characteristics, IEDDA can proceed in low concentrations of reactants. Using IEDDA, various dienes such as tetrazines as a non-limiting example and dienophiles such as trans-cyclooctene and norbornenes, as non-limiting examples, can be mixed in suitable ratios to form conjugates. The reaction proceeds at rapid rates without the use of catalysts or any other additives in different solvents such as dichloromethane, acetonitrile, tetrahydrofuran, methanol and ethanol as non-limiting examples.

[0014] Polymers that can be used include the polyesters such as polylactide, polycaprolactone, polylactide-co-glycolide, copolymers of the polyesters with polyethylene glycol, other copolymers, or any other polymer or copolymer that can be functionalized to bear dienes and dienophiles to facilitate the IEDDA reaction or with other functional groups to facilitate polymer coupling. Using this approach, the choice of solvent used must be such that the different polymers are initially soluble in the solvent but upon conjugation, the dipolymer becomes insoluble in the solvent. A combination of solvents could also be used such that a non-solvent for the dipolymer (e.g. water, alcohols) may be combined with the solvent for the constituent polymers to facilitate nanoparticle precipitation.

[0015] As an example of the present method, commercially available HO-PLGA-PEG (HO-poly (lactide-co-glycolide)-polyethylene glycol) or HO-PLGA (HO-poly (lactide-co-glycolide) is functionalized with trans-cyclooctene (TCO; a dienophile) to yield TCO-PLGA-PEG or TCO-PLGA respectively. Another commercially available HO-PLGA is functionalized with tetrazines (such as methyltetrazine, MTZ) to yield Tetrazine-PLGA. The reaction of these functionalized PLGA molecules (TCO-PLGA-PEG or TCO-PLGA and Tetrazine-PLGA) that is dissolved and soluble in a rationally selected combination of solvents leads to a polymer dimer with double the molecular weight and thus insoluble in the original solvent mixture. This leads to precipitation of the polymer and its assembly to form nanoparticles based on the composition of the initial solvent mixture. If there is water in the solvent mixture for example, the PEG molecules orient into the aqueous phase forming the corona of the nanoparticle while the PLGA forms the core leading to the formation of a PEG-coated stealth nanoparticle capable of prolonged circulation in blood. Since only a dimer is formed (unlike polydisperse polymers and oligomers in dispersion polymerization), relatively precise insoluble polymer chain lengths are formed which leads to relatively precise particle sizes with very narrow size distribution. Also, there is no particle growth as obtained in dispersion polymerization and other polymerization methods of nanoparticle fabrication leading to narrow particle size distribution. In addition, the PEG at the surface acts as a steric stabilizer and prevents coagulation of particles thereby controlling the particle size in addition to conferring stealth properties to the nanoparticle in vivo.

[0016] Other non-limiting examples of the present method is the reaction between TCO-PEG and MTZ-PLGA (Methyltetrazine-PLGA) or TCO-PLGA and MTZ-PEG of suitable molecular weights in a suitable solvent composition. Additional examples include the reaction between TCO-PLGA-PEG and MTZ-PLGA-PEG. The molecular weight of polymers to be functionalized with TCO-(trans-cyclooctene) or MTZ-(methyltetrazine), and the solvent composition for the reaction among other formulation variables are essential in this approach and must be optimized.

[0017] While the subject matter disclosed herein has been described in connection with what is presently considered to be practical example embodiments, it is to be understood that the present disclosure is not limited to the disclosed embodiments, and covers various modifications and equivalent arrangements included within the spirit and scope of the present invention.