**1. Introduction**

With the first clinical approval of a polymer-based nano-drug in 1995 [1], interest in developing polymers as nanocarriers of (bio)pharmaceutical drugs has been steadily growing [2,3]. Due to their favorable characteristics, e.g., adjustable physical and mechanical properties, it is not surprising that polymer-based nanomaterials are now established in many areas of bionanotechnology. Polymers are widely used in delivery systems for therapeutics, in matrices for tissue engineering and, among others, in polymer-based composites for biomedical purposes [4–6].

The main criteria in designing polymers for therapeutic use have been based mainly on the biocompatibility and the biodegradability of the polymer backbone as well as the suitability of the polymer to be processed into a stable pharmaceutical formulation [7].

**Citation:** Vollrath, A.; Kretzer, C.; Beringer-Siemers, B.; Shkodra, B.; Czaplewska, J.A.; Bandelli, D.; Stumpf, S.; Hoeppener, S.; Weber, C.; Werz, O.; et al. Effect of Crystallinity on the Properties of Polycaprolactone Nanoparticles Containing the Dual FLAP/mPEGS-1 Inhibitor BRP-187. *Polymers* **2021**, *13*, 2557. https:// doi.org/10.3390/polym13152557

Academic Editors: José Miguel Ferri, Vicent Fombuena Borràs and Miguel Fernando Aldás Carrasco

Received: 29 June 2021 Accepted: 29 July 2021 Published: 31 July 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

However, there are other parameters of equal importance to be considered to optimize a polymer for its application as a delivery vehicle. In fact, parameters such as molar mass, functional end-groups, hydrophobic-hydrophilic balance (HHB), melting temperature (Tm) and crystallinity strongly influence the drug loading and the drug release kinetics from the polymer matrix [8].

Independent investigations of the influence of the polymer crystallinity on the resulting particle characteristics (e.g., particle formation and degradation) while keeping the key properties of the system constant are rare or only provide partial conclusions typically due to influences of a third variable [9]. In particular, alterations of HHB are frequently accompanied by changes in crystallinity [10]. It is hence currently not fully understood if degradation or general performance of hydrophobic pharmapolymers in aqueous media are, in fact, strongly influenced by polymer crystallinity or if the hydrophobicity is the dominating factor.

Today, the most commonly used polymers for biomedical applications are polyesters, such as polylactide (PLA), poly(lactide-*co*-glycolide) (PLGA) and polycaprolactone (PCL) [11]. They are easy to access and offer a range of interesting advantages; i.e., (i) a complete hydrolytic and/or enzymatic biodegradation, (ii) a facile and controlled synthesis to obtain defined molar masses, (iii) various modification possibilities of the polymer structure, and (iv) commercial availability [12]. The advantages of PCL compared to other aliphatic polyesters include interesting thermal properties, higher durability and manufacturability, and a good compatibility with other polymers [13,14]. Thus, PCL represents a promising candidate to design materials with tailor-made properties [13,15,16]. Bandelli et al. recently demonstrated that copolymerization of the constitutional isomers ε-caprolactone (εCL) and δ-caprolactone (δCL) with a varying ratio of εCL and δCL can generate a library of five copolyesters featuring a constant HHB and similar molar masses in the range of 7 to 10 kDa, but the copolymers showed a varying crystallinity [17]. They are hence suitable materials to study the sole influence of crystallinity on the particle properties and performance. In this study, we utilized this library of poly(εCL-*ran*-δCL) to formulate drug-loaded nanoparticles (NPs). The aim was to investigate, firstly, whether such polymers provide suitable properties (particle size and polydispersity) to form an NP-based drug delivery system, and secondly, to study the effect of the crystallinity of the bulk polymers on the enzymatic degradation and the in vitro performance of the resulting NPs.

The anti-inflammatory drug BRP-187 (4-(4-chlorophenyl)-5-[4-(quinoline-2-ylmethoxy) phenyl] isoxazol-3-carboxylic acid) is a dual inhibitor of the 5-lipoxygenase-activating protein (FLAP) and microsomal prostaglandin E2 synthase-1 (mPGES-1), which are crucial proteins within arachidonic acid (AA) metabolism. Inhibition of mPGES-1 and FLAP prevents the biosynthesis of pro-inflammatory prostaglandin (PG)E<sup>2</sup> and leukotrienes (LTs), respectively [18]. Several in vitro and in vivo studies with inhibitors of FLAP and/or mPGES-1 have demonstrated their efficient anti-inflammatory activity while exhibiting fewer adverse effects compared to the conventional non-steroidal anti-inflammatory drugs (NSAIDs) [19,20]. These observations suggest that dual inhibition of FLAP and/or mPGES-1, rather than blocking cyclooxygenase-1 or -2 pathways, might be a better strategy for intervention with inflammation. However, BRP-187 is a fatty acid-like molecule with poor water solubility and a strong tendency to bind plasma proteins [18]. Molecules exhibiting such properties typically cause challenges in reaching a sufficient bioavailability in vivo and require technological solutions to improve their pharmacokinetic drawbacks. We have previously demonstrated that encapsulating BRP-187 into PLGA NPs and acetalated dextran NPs enhanced its enzyme inhibition efficacy in vitro [21].

In the present study, we initially performed a high-throughput (HT) nanoprecipitation approach for the homopolymers PεCL and PδCL to screen a range of polymer and surfactant concentrations for the preparation of empty (unloaded) PCL particles. Once optimal formulation conditions were established, and drug-loaded NPs were prepared by batch nanoprecipitation of the PεCL and PδCL homopolymers as well as of the poly(εCL-*ran*δCL) copolymers with BRP-187. PCL NPs with and without BRP-187 were characterized

for their critical quality attributes, namely their particle size, polydispersity index (PDI), surface charge and drug loading. Other particle properties i.e., nanodispersion stability and NP degradation behavior were also investigated. Ultimately, BRP-187-NPs were studied in vitro in human polymorphonuclear leukocytes (PMNL) for their efficiency to inhibit the drug target FLAP, in comparison to the free BRP-187.
