MARIE SKŁODOWSKA-CURIE ACTIONS Individual Fellowships (IF) H2020-MSCA-IF-2018

Phase-separated block copolymer nanoparticles for spatial organization of enzymes:
A new strategy to control enzyme cascade reactions


Acronym: emzymeCOMP            Project ID: 838448           April 2019 - March 2021


Dr. Lucila Navarro


Summary of the context and overall objectives of the project:

Nature has developed a unique set of mechanisms to control biological functions through compartmentalization of functional macromolecules such as enzymes. Inspired by this high level of control, there is an increasing interest in mimicking such accurate spatial separation on synthetic colloidal materials. However, most current approaches show limited spatial control over chemical functionalities. Consequently, new strategies are needed to localize functional (macro)molecules in colloidal materials with more precision.


   To address this need, the phase-separation of block copolymers (BCP) in colloidal confinements is a promising strategy to generate particles with well-defined chemical patterns. Especially, striped ellipsoidal particles are of interest due to their well-defined lamellar pattern of alternating chemical domains on the particle surface. Thus, the development of domain-specific functionalization strategies would open up new biomimetic applications such as compartmentalization of cascade enzymes.


   Working towards this vision, different strategies have been reported but either focus on the functionalization of the particle interior or are limited to the functionalization of a single domain. Thus, this project developed two different strategies to overcome current shortcomings in domain-specific surface functionalization of striped ellipsoidal BCP particles:


(1)  Surface functionalization through external surfactants: Two different block-selective surfactants are designed to interact each with a specific block of the structure-forming BCP during the particle preparation. This results in patterned particle surfaces where the functionality of the stripes is now determined by orthogonally reactive end-groups of the surfactants.


(2) Surface functionalization through internal domains. Reactive monomers are incorporated by domain-specific swelling after the particle formation. Subsequent polymerization introduces additional reactivity into the different domains. This translates from the domain interior to the surface of the particles, which allows further domain-specific surface functionalization.


Detailed investigations on both strategies revealed the importance of fundamental examinations to get a more thorough understanding of the underlying principles and select the most promising strategy. A careful comparison demonstrated the synthetic flexibility and versatility of approach (2), which introduces the reactivity after the particle preparation. Thus, established routes to striped ellipsoids can be used to circumvent elaborate optimizations of phase-separation conditions, which are needed in strategy (1). In addition, strategy (2) enables the preparation of novel anisotropic lamellar thicknesses, which enables a new level of spatial control over the chemical functionality.


Work performed from the beginning of the project to the end of the period:

To enable the surface functionalization through functional surfactants, two different surfactant molecules were synthesized as amphiphilic block copolymers containing reactive end-groups. An alkyne group was introduced for alkyne-azide “click” functionalization of the particle surface. Functionalization of the second surfactant with an orthogonally reactive succinimide group was started but then delayed due to shut down research facilities in course of the COVID-19 pandemic. Overall, this strategy successfully demonstrated the formation of striped ellipsoidal BCP particles by using tailormade surfactants, thus implementing the crucially needed expertise to further develop this approach in follow-up projects.


   To enable surface functionalization through modified internal domains, reactive monomers were used for a seeded polymerization with striped ellipsoidal particles. Key to this approach is a two-step process: First, monomers selectively swell one BCP domain. Second, polymerization covalently fixates the morphology. It was found that selective swelling leads to polymer chain stretching which causes restructuring of the particles. This can be used to control the thickness of one specific domain, which creates a previously unknown internal anisotropy and translates into highly elongated particles. Based on these fundamental insights, a reactive monomer was selectively incorporated into one domain to enable spatially controlled modification via click reactions. As proof of concept, coupling of dyes was shown by UV spectroscopy and the local functionalization with ferrocene was demonstrated by energy-dispersive X-ray spectroscopy (EDX).


   The simplicity and the robustness of this approach represents a unique new tool to control the lamellae thickness and aspect ratio of striped ellipsoidal particles. As a post-particle formation approach, this functionalization allows obtaining a variety of morphologies from one batch of particles. The addition of reactive functional groups through monomers lays the foundation for further functionalization that can ultimately be used to mimic cellular compartmentalization.


    The results of both projects were presented at the 16th ZSIGMONDY COLLOQUIUM 2020 "Soft Colloids" in Dusseldorf, Germany and highlighted with the Best Poster Award. 

Navarro, L., Klinger, D. From Striped Ellipsoids to Janus Nanoparticles: Controlling Colloid Shape, Morphology and Function via Phase Separation of Functional Polymers. 16th ZSIGMONDY COLLOQUIUM 2020 "Soft Colloids". Dusseldorf, Germany. 2020


   Unfortunately, other disseminations on international congresses organized for the year 2020 were fully postponed because of the COVID-19 pandemic. This includes the American Chemical Society (ACS) meeting and the Makromolekulares Colloquium in Freiburg, Germany.


   The results from the project will be published open access in two main articles regarding the swelling-induced restructuring and the seeded-polymerization (manuscripts are in preparation). In addition, a review article was published to highlight the potential of such particle-functionalization approaches:


   Gruber, A., Navarro, L., Klinger, D., Reactive Precursor Particles as Synthetic Platform for the Generation of Functional Nanoparticles, Nanogels, and Microgels. Adv. Mater. Interfaces 2020, 7, 1901676.


Progress beyond the state of the art, expected results until the end of the project:

In this project, two different strategies were followed to enable the site-specific surface functionalization of striped ellipsoidal BCP nanoparticles. Overall, the results contribute to a more thorough understanding of the colloidal chemistry and physics of phase-separated BCP nanoparticles. On one hand, the potential of tailor-made surfactants was demonstrated and builds the foundation for a synthetic platform for surface functionalization. On the other hand, investigations on particle swelling and seeded polymerization lead to a new understanding of the solvent-dependent morphological restructuring. This provides a new foundation to modify the particles’ colloidal features (morphology and shape) after their preparation. In addition, the selective incorporation of reactive groups in specific lamellae creates reactive domains that could have tremendous implications in the bio-chemical field. Especially the utilization as a new synthetic platform to study enzyme compartmentalization could ultimately lead to new technologies, e.g., new catalytic systems.and potential impacts.