**1. Introduction**

Bacterial infections are the second leading cause of death worldwide with an estimated 7.7 million deaths annually, of which 5 million are associated with antimicrobial resistance (AMR) to existing antibiotics [1]. They are a constant threat to global health systems and a continuing threat to human lives. Certainly, highly effective therapeutic interventions are needed more than ever to target bacterial infections and offset the rise in AMR. While various innovative approaches to discovering and developing new antibacterial agents have been proposed or undertaken such as the use of antibodies [2], aptamers [3], nucleic acid materials [4], nanoparticles of metal oxides [5–7], small synthetic chemical compounds [8,9], and others [10], antimicrobial host defense peptides (HDPs), which are part of the innate immune system, have been proven as a promising potential therapeutic strategy to confront bacterial infections and tackle the clinical threats of biofilm-forming drug-resistant strains due to the multiple advantages they offer [11]. HDPs are relatively small polymers (usually, 4–50 amino acid residues) that can be easily synthesized and modified at minimal costs. Unlike antibodies and small chemical compounds of nucleic acid-based drugs, antimicrobial HDPs often exhibit broad-spectrum activity against a wide range of bacteria species, including Gram-positive and Gram-negative pathogens, and usually, they tend to have a low propensity to develop antimicrobial resistance due to their multifaceted mechanism of actions [12,13]. These HDPs are also biodegradable, and tend to degrade easily; thus, they do not persist in the body and do not pose any undesirable side effects [13]. Furthermore, the ability of these antimicrobial peptides to disperse and eradicate mature bacterial

**Citation:** Etayash, H.; Yip, F.; Hancock, R.E.W. Impacts of PEGylation and Glycosylation on the Biological Properties of Host Defense Peptide IDR1018. *Pharmaceutics* **2023**, *15*, 1391. https://doi.org/10.3390/ pharmaceutics15051391

Academic Editors: Scavello Francesco, Amiche Mohamed and Jean-Eric Ghia

Received: 17 March 2023 Revised: 18 April 2023 Accepted: 27 April 2023 Published: 1 May 2023

**Copyright:** © 2023 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/).

biofilms and high-density bacterial infections both in vitro and in vivo gives them advantages over current antibiotics and other molecules as excellent antimicrobial and antibiofilm drug candidates [11]. Also, the ability of HDPs to act as anti-infective immune modulators with promising anti-inflammatory activity makes them clinically attractive candidates for multipurpose applications [14,15]. However, despite their advantages, as pharmaceutical products, HDPs endure several constraints that limit their ease of accessibility and use, including their systemic aggregation and toxic side effects typically at higher doses, in addition to their poor stability against blood-borne proteases [13,16]. While various approaches including, for instance, formulation strategies, sequence modifications and designing HDP mimetics have been explored to circumvent some of these shortcomings of HDPs, only limited successes for different peptides were achieved [13,17–23]. In this study, we decided to explore the special effects of both pegylation and glycosylation on the physiochemical properties as well as the biological activity properties of the HDP, IDR1018, which is a synthetic multifaceted peptide with immunomodulatory and antibiofilm activities [24,25]. Pegylation and glycosylation are modifications utilized in peptide/protein drug design and are often used to improve the stability and pharmacokinetic assets of potential drug candidates while reducing toxicities and potentially harmful side effects [26–29]. Pegylation is a process by which peptides are chemically conjugated to polyethylene glycol (PEG) in order to change their physicochemical or biological properties [30]. As PEG and its derivatives are inert, water-soluble, non-toxic and non-immunogenic, they are widely used to overcome limitations associated with biopharmaceutical products including water solubility issues, aggregations, toxicity against mammalian cells and immunogenicity [30]. Pegylation offers significant advantages for biopharmaceutical products, as attaching PEG improves proteolytic stability, helps mitigate the immunogenicity, increases resistance to bacterial-secreted enzymes, boosts blood circulation half-lives and enhances biodistribution as well as drug bioavailability [28,30]. Nevertheless, despite these advantages, pegylation is often associated with a partial or complete reduction in the antimicrobial activity of HDPs [30]. Glycosylation is a process by which a sugar moiety is chemically attached to biopharmaceutical molecules such as peptides, proteins, antibodies, etc., in order to change their physicochemical properties or produce better bioactive compounds [31]. As with pegylation, glycosylation can have a significant influence on the properties of HDPs, for example, modifying toxicities, resistance to proteolytic degradation, pharmacokinetics and dynamic properties [27,31,32]. However, as with pegylation, glycosylation does not always improve the antibacterial activity of HDPs since the attaching surges change the chemical structure, hydrophobicity and overall charge of the peptides, which can impact the insertion and interaction of peptides with bacterial membranes [31].

Indeed, the overall influence of pegylation and glycosylation on drug design is remarkable with many clinically accepted drugs being pegylated and glycosylated. Since little is known about the influence of both conjugations on the multifaceted properties of HDPs, especially the immunomodulatory functionality, we aimed in this study to look at their overall impacts by covalently modifying the antimicrobial HDP IDR1018 with short-chain PEG (PEG6) and a glucose moiety (N-acetyl glucosamine (GlcNAc)).

### **2. Materials and Methods**

#### *2.1. Peptide Synthesis, Pegylation, and Glycosylation*

IDR1018 peptide (sequence, VRLIVAVRIWRR-NH2) was purchased from GenScript (Piscataway, NJ, USA) at >95% purity. The pegylated IDR1018 [PEG6; VRLIVAVRIWRR-NH2] as well as the glycosylated IDR1018 [Glc-IDR1018; T(GlcNAc)VRLIVAVRIWRR-NH2], were obtained at ≥95% purity from Biomatik LLC (Wilmington, DE, USA) (Table 1).


**Table 1.** IDR1018 and its conjugated derivatives, HPLC, and mass spectra data.

HPLC, high-performance liquid chromatography; Ret. Time, retention time in minutes; MW, molecular weight; Calc., calculated; Obs., observed.

### *2.2. Aggregation Assay*

Solutions of IDR1018 peptide and the conjugates were tested for aggregation in saline (0.9% NaCl), 5% dextrose, and 10% RPMI tissue culture medium at 1 mg mL−1. The solutions were placed in 96 well plates and sterile water was used as a negative control. The turbidity was determined for each sample as described in earlier reports [19,33]. Similarly, the conjugates were screened for aggregation in the presence of sodium salts of polyatomic anions (citrate or phosphate) at various strengths (0.1−1000 mM). The % of aggregates was assessed relative to the OD600 of sterile water.

Furthermore, isolated peripheral blood mononuclear cells (PBMCs) seeded in 96-well flat-bottom tissue culture plates (Corning Inc., Corning, NY, USA) at a concentration of <sup>2</sup> × <sup>10</sup><sup>6</sup> in RMPI media were treated with the two conjugates at a final concentration of 32 μg mL<sup>−</sup>1, incubated for 4 h at 37 ◦C in 5% CO2, and visualized on a Nikon Eclipse TS100 microscope. Experiments were performed in triplicate in three independent experiments. Representative images of the microscopy are presented in Figure 1.
