Advanced Vaginal Nanodelivery of Losartan Potassium via PEGylated Zein Nanoparticles for Methicillin-Resistant Staphylococcus aureus

Abstract

Background/Objectives: PEGylated zein nanoparticles (PZNs) loaded with losartan potassium (LOS) were developed as a repurposed treatment for vaginal methicillin-resistant Staphylococcus aureus (MRSA) infection. PZNs were prepared using the ethanol injection method with different types and amounts of Brij^® surfactant.

Methods: The prepared formulations were optimized using a D-optimal mixture design via Design-Expert^® software version 13. The assessed responses included entrapment efficiency (EE%), particle size (PS), and zeta potential (ZP).

Results: The optimized PZNs, composed of 30 mg Brij^® O20 and 10 mg zein, exhibited spherical particles with an EE% of 90.58 ± 1.20%, PS of 200.81 ± 1.39 nm, PDI of 0.395 ± 0.01, and ZP of −36.59 ± 0.05 mV. Confocal laser scanning microscopy confirmed complete deposition of fluorescein-labeled PZNs within vaginal tissues. Ex vivo studies showed that PZNs resulted in prolonged permeation of LOS compared to the LOS solution. In a murine model of MRSA infection, the optimized PZNs demonstrated superior therapeutic efficacy over the LOS solution. Histopathological examinations confirmed the safety of the tested formulations.

Conclusions: In conclusion, the optimized PZNs present a promising approach for the treatment of MRSA-related vaginal infections.

Introduction

Infections caused by antimicrobial-resistant bacteria represent a growing global public health threat [1]. Pathogens often colonize the birth canal via fecal contamination [2], and can be transmitted to the newborn during labor, particularly following prolonged or obstructed labor or premature rupture of membranes, making neonatal bacterial sepsis a leading cause of morbidity in the first week of life [3]. Methicillin-resistant Staphylococcus aureus (MRSA) is traditionally associated with nosocomial outbreaks but has also emerged in community settings. These infections can be severe, prolong hospital stays, and carry high mortality rates. MRSA is mainly transmitted by transiently contaminated hands of healthcare workers or through direct contact with infected lesions and contaminated surfaces. Common carriage sites include the anterior nares and vagina, with the perineum also frequently colonized; less common reservoirs include the throat and axillae [4]. Vaginitis, a symptomatic inflammation of the vagina characterized by discharge, itching, and pain, can both result from and facilitate MRSA colonization, underscoring the need for targeted preventive and therapeutic strategies [5].

MRSA is an opportunistic commensal that can cause a spectrum of diseases, from mild skin and soft-tissue infections to severe, life-threatening conditions, such as pneumonia, endocarditis, sepsis, and toxic shock syndrome. Its clinical impact has intensified due to limited therapeutic options against multidrug-resistant strains and biofilm-associated infections [6,7]. Repurposing non-antibiotic drugs with demonstrated in vitro antibacterial activity offers a promising strategy to address this challenge [8]. Drug repurposing involves using an existing drug for new indications beyond its original approval. This approach benefits from extensive pre-existing pharmacokinetic and safety data, which helps shorten development time [9]. Notably, antipsychotics like promazine have shown activity against MRSA, Klebsiella pneumoniae, and other pathogens by disrupting membrane integrity [10], while statins (antihyperlipidemics) can impair Staphylococcus aureus and Streptococcus pneumoniae viability by promoting membrane dysfunction, apoptosis, and inhibiting protein synthesis [8].

MRSA can survive intracellularly, and the success of antimicrobial therapy depends on how effectively drugs penetrate infected tissues and persist within cells. A drug’s ability to cross cellular membranes and remain intracellularly defines its therapeutic concentration at the infection site. Nanocarriers (NCs) enhance drug permeation and accumulation. Their direct interaction with MRSA, along with the subsequent release of the encapsulated drug, facilitates improved uptake and sustained antibacterial action [11]. Among various NC systems, zein-based nanoparticles (ZNs) are especially promising. Zein, a naturally occurring, hydrophobic prolamin protein [12], serves as a biocompatible carrier that also contributes intrinsic antimicrobial effects [13,14].

ZNs have attracted considerable attention for their biocompatibility, biodegradability, processability, and ability to encapsulate both hydrophilic and hydrophobic drugs, all while offering exceptional colloidal stability [12]. Their high surface-to-volume ratio and nanoscale dimensions enhance interactions with biological fluids and promote efficient cellular uptake of loaded drugs. Prior studies have demonstrated the antimicrobial efficacy of ZNs against Pseudomonas aeruginosa and Streptococcus mutans [13,14]. In this work, we employed Brij®, a PEGylated single-chain edge activator with variable PEG lengths and fatty-acid moieties, to formulate PEGylated zein nanoparticles (PZNs). Polyethylene glycol (PEG) is an uncharged, hydrophilic polymer widely used in pharmaceutical formulations, particularly for topical and vaginal applications. PEGylation enhances the interaction of nanoparticles with vaginal mucus and epithelium, improving drug distribution, retention, and therapeutic efficacy. The combination of PEG’s mucus-penetrating properties with the protective nature of nanocarriers enables controlled and localized drug release near the vaginal epithelium [15,16].

To our knowledge, there have been no studies examining PZNs as an advanced drug delivery system for repurposed losartan potassium (LOS), which is a previously known antihypertensive drug, in the treatment of vaginal MRSA infections. Therefore, this study aimed to optimize PZNs to enhance LOS vaginal distribution and retention and to evaluate their safety and efficacy. Using a D-optimal mixture design, we investigated three formulation factors: Brij® amount (X1), zein amount (X2), and Brij® type (X3), defined as critical process parameters (CPPs), and measured their effects on entrapment efficiency (EE%; Y1), particle size (PS; Y2), and zeta potential (ZP; Y3), which represent the Critical Quality Attributes (CQAs). We also assessed the effect of storage over time. Ex vivo permeation studies compared LOS solution versus optimized PZNs, and confocal microscopy tracked fluorescein-labeled PZN deposition within vaginal tissues. In vitro assays evaluated antibacterial activity and in vivo experiments in a murine model assessed therapeutic efficacy and safety of the formulations.

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Materials

Losartan potassium (LOS) was obtained from the Egyptian International Pharmaceutical Industries (EIPICO; Cairo, Egypt). Brij® O20, Brij® 93, L-α-phosphatidylcholine (PC), fluorescein diacetate (FDA), and zein were purchased from Sigma Aldrich (St. Louis, MO, USA). Methanol and ethanol were obtained from El-Nasr Pharmaceutical Chemicals Company (Abu Zabal, Egypt). All other chemicals and reagents used were of analytical grade.

Albash, R.; Hassan, M.; Agiba, A.M.; Mohamed, H.W.; Hassan, M.S.; Ali, R.M.; Shalabi, Y.E.; Omran, H.M.A.; Eltabeeb, M.A.; Alamoudi, J.A.; et al. Advanced Vaginal Nanodelivery of Losartan Potassium via PEGylated Zein Nanoparticles for Methicillin-Resistant Staphylococcus aureus. Pharmaceutics 2025, 17, 1344. https://doi.org/10.3390/pharmaceutics17101344