Author: Sukhachov Denys Pavlovych

ABSTRACT

Background. The problem of antibiotic resistance has reached the scale of a global crisis: annually, more than one million patients worldwide die from complications caused by drug-resistant infections . The rise of multidrug-resistant strains, particularly methicillin-resistant Staphylococcus aureus (MRSA) and Klebsiella pneumoniae, necessitates the search for fundamentally new approaches to antibacterial therapy that would combine different mechanisms of action and prevent the rapid formation of resistance . A promising direction is the creation of hybrid nanocomposites that unite several antibacterial agents within a single delivery system.

Objective. Theoretical substantiation of the design, development of a synthesis strategy, and analysis of the prospects of a triple hybrid system based on moxifloxacin, tetrahydrocannabinol (THC), and silver nanoparticles functionalized with β-cyclodextrin, utilizing a redox-sensitive disulfide linker for intracellular component release.

Materials and Methods. An analysis of current literature data on the antibacterial properties of fluoroquinolones, cannabinoids, and silver nanoparticles was conducted; the mechanisms of synergistic action of combined systems were investigated; approaches to creating “guest-host” inclusion complexes using β-cyclodextrin were analyzed; types of biodegradable linkers for controlled drug release were reviewed.

Results. The architecture of the hybrid nanocomposite Moxifloxacin—S—S—THC ⊆ β-Cyclodextrin—AgNP is proposed. The selection of moxifloxacin as the fluoroquinolone with the broadest spectrum of action, covering gram-positive, gram-negative bacteria, and anaerobes, as well as possessing high lipophilicity and the ability to penetrate inside cells, is justified. It is shown that β-cyclodextrin performs a dual function: reducing silver ions to metallic Ag⁰, stabilizing nanoparticles (size 3-20 nm), and forming an inclusion complex with the hydrophobic THC molecule . The use of a disulfide linker to connect moxifloxacin to THC is proposed; this linker is cleaved by high concentrations of glutathione inside the bacterial cell, ensuring the release of the fluoroquinolone to reach its target (DNA gyrase/topoisomerase IV). The prospects for the synergistic action of the three components are discussed: inhibition of DNA synthesis, disruption of the cell membrane, and induction of oxidative stress.

Conclusions. The developed design of the hybrid nanocomposite opens new possibilities for overcoming antibiotic resistance due to the combination of three different mechanisms of antibacterial action, which prevents the rapid formation of resistance in pathogenic microorganisms. The proposed system represents a promising platform for further experimental research.

Keywords: antibiotic resistance, fluoroquinolones, moxifloxacin, tetrahydrocannabinol, silver nanoparticles, β-cyclodextrin, disulfide linker, hybrid nanocomposites, synergism.

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1. INTRODUCTION

1.1. The Problem of Antibiotic Resistance and the Search for New Strategies

Antibiotic resistance has been recognized by the World Health Organization as one of the greatest threats to global health, food security, and development. The frequency of infections caused by antibiotic-resistant bacteria is increasing worldwide daily, raising healthcare costs, morbidity, and mortality . According to research, more than one million patients die annually from complications caused by drug-resistant infections, and this number is only expected to grow .

The main factors leading to increased antimicrobial resistance are inadequate infection control and incorrect antibiotic use. In approximately 50% of cases, antibiotic use is irrational, creating significant selective pressure and contributing to the emergence of resistant pathogens . Particularly dangerous are multidrug-resistant hospital infections, such as Klebsiella pneumoniae (Friedländer’s bacillus), which are extremely toxic and insensitive to almost all existing antibiotics .

As noted by M. Bassetti (2015), we are on a dead-end path: bacteria adapt to antibiotic therapy faster than new drugs are developed. Medications with fundamentally new mechanisms of action are needed . A promising direction is the creation of combined preparations that affect the bacterium simultaneously from different sides. Research from the Institute of Molecular Biology and Genetics of the National Academy of Sciences of Ukraine has shown that combining non-toxic antibiotics, each individually ineffective, can yield excellent results due to synergistic action .

1.2. The Concept of Hybrid Nanocomposites

One of the most promising approaches to overcoming antibiotic resistance is the creation of hybrid nanocomposites that combine several antibacterial agents within a single delivery system. Silver nanoparticles (AgNPs) attract particular attention due to their unique physicochemical and bioactive properties. Silver is a well-known antimicrobial agent effective against bacteria, viruses, fungi, and yeasts, including several antibiotic-resistant strains .

The biochemical properties of AgNPs depend on various parameters, including size, shape, and stability, which are determined by the methodology and conditions of their preparation. The use of nanoparticles significantly enhances these properties due to the dramatic increase in surface area .

1.3. Objective of the Study

The aim of this work is the theoretical substantiation of the design of a triple hybrid system based on moxifloxacin, tetrahydrocannabinol (THC), and silver nanoparticles functionalized with β-cyclodextrin, using a redox-sensitive disulfide linker that ensures the intracellular release of components.

2. CHARACTERISTICS OF THE HYBRID SYSTEM COMPONENTS

2.1. Fluoroquinolones: Moxifloxacin as the Optimal Choice

Fluoroquinolones are broad-spectrum bactericidal antibiotics that penetrate the bacterial cell and block two key enzymes—DNA gyrase and topoisomerase IV—halting DNA synthesis. They are active against gram-negative (Escherichia coli, Pseudomonas aeruginosa), gram-positive (staphylococci), and atypical pathogens (Chlamydia, Mycoplasma) .

Moxifloxacin was chosen for the proposed hybrid system for the following reasons:

• Broadest spectrum of action: covers gram-positive, gram-negative bacteria, anaerobes, and atypical pathogens
• High lipophilicity: ensures excellent penetration inside cells, critical for the intracellular release of components
• Convenient chemical structure: presence of functional groups for linker attachment
• Presence of a fluorine atom: fluorine is already incorporated into the molecule’s structure at the 6-position, a structural feature of this antibiotic class

It is important to note that some fluoroquinolones have serious side effects: tendon damage, cardiotoxicity (QT interval prolongation), toxicity to the central nervous system, and liver. Moxifloxacin demonstrates a moderate toxicity profile compared to other class representatives, making it a better candidate for inclusion in the hybrid system.

2.2. Tetrahydrocannabinol: Antibacterial Properties and Limitations

Recent studies confirm that cannabinoids, including THC, possess antibacterial properties:

• Spectrum of action: THC is active primarily against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). The minimum inhibitory concentration for staphylococci and streptococci is in the range of 1–5 μg/mL.
• Limitation: Against gram-negative bacteria, THC alone is ineffective due to the presence of an additional outer membrane that hinders molecule penetration.
• Mechanism of action: THC disrupts the bacterial cell membrane, compromises its integrity, leading to a decrease in ATP levels (energy resource of the cell) and an increase in reactive oxygen species.
• Against biofilms: THC can disrupt MRSA biofilms, which is critically important because biofilms make bacteria extremely resistant to treatment.

A significant limitation is that in the presence of 4% serum or 5% blood, THC activity markedly decreases, which may pose a challenge for systemic application.

2.3. Silver Nanoparticles and β-Cyclodextrin

Cyclodextrins (α-, β-, and γ-CD) are natural cyclic oligosaccharides formed during starch degradation. They have a cone shape with a hydrophobic internal cavity and a hydrophilic outer surface, ensuring their high water solubility .

β-Cyclodextrin holds a middle position due to its moderate cavity size (0.6 nm) compared to α- and γ-cyclodextrins . It can serve as an effective reagent for reducing metal salts and can bind to the nanoparticle surface through chemisorption, playing an important role in preventing nanoparticle aggregation and promoting their stability in solution .

Studies from 2021-2025 confirm that β-cyclodextrin-functionalized silver nanoparticles (β-CD-AgNPs) can be obtained with particle sizes of 3-20 nm and high dispersity . They exhibit a characteristic plasmon band at wavelengths of 423-433 nm, confirming the formation of spherical nanoparticles .

3. DESIGN OF THE HYBRID NANOCOMPOSITE

3.1. General Architecture

The proposed hybrid system has the following architecture:

Moxifloxacin — S—S — THC ⊆ β-Cyclodextrin—AgNP

Where:

• Moxifloxacin — S—S — THC: a disulfide linker that is cleaved inside the bacterial cell
• ⊆ — inclusion complex of THC in the β-cyclodextrin cavity
• AgNP — silver nanoparticle stabilized by β-cyclodextrin

3.2. Functional Components and Their Roles

Table 1. Components of the Hybrid System and Their Functions

Component	Primary Function	Mechanism of Action
Moxifloxacin	DNA synthesis inhibition	Blockade of DNA gyrase and topoisomerase IV
THC	Cell membrane disruption	Destabilization of lipid bilayer, ATP depletion, ROS increase
AgNP	Oxidative stress, membrane damage	ROS generation, interaction with protein thiol groups
β-Cyclodextrin	AgNP stabilization, complexation with THC	Formation of “guest-host” complex
Disulfide linker	Controlled release	Cleavage by intracellular glutathione

3.3. The Role of β-Cyclodextrin in the System

β-Cyclodextrin performs a dual function in the proposed system:

1. Stabilization of silver nanoparticles: β-CD is used as a reducing agent and stabilizer in the preparation of silver nanoparticles. It reduces Ag⁺ ions to metallic Ag⁰ and prevents the aggregation of the formed nanoparticles . The obtained β-CD-AgNPs have a size in the range of 3-20 nm and maintain stability over extended periods .
2. Formation of an inclusion complex with THC: Due to its hydrophobic cavity, β-cyclodextrin can include hydrophobic THC molecules, forming a stable “guest-host” complex. This solves the problem of low THC water solubility and ensures its retention on the nanoparticle surface.

Studies using triclosan as an example have shown that successful inclusion complex formation is confirmed by the disappearance of the characteristic endothermic peak of the guest molecule in differential scanning calorimetry (DSC) curves . A similar approach can be applied to confirm complex formation between β-CD-AgNPs and THC.

4. LINKER TECHNOLOGY: DISULFIDE LINKER

4.1. Principle of Disulfide Linker Action

Disulfide linkers are the “gold standard” for intracellular drug release. They are cleaved by glutathione—a molecule whose concentration inside cells is 100–1000 times higher than in the extracellular environment.

The principle of action is as follows:

1. The hybrid molecule enters the bacterial cell
2. High glutathione levels reduce the disulfide bond (-S-S- → -SH HS-)
3. The linker breaks, releasing moxifloxacin
4. Free moxifloxacin reaches its target (DNA gyrase/topoisomerase IV)
5. THC remains bound to the silver nanoparticle via the β-cyclodextrin complex

4.2. Advantages of the Disulfide Linker

The disulfide linker was chosen for the following reasons:

• Selectivity: cleaved exclusively inside the cell, minimizing premature moxifloxacin release in the bloodstream
• Well-studied: used in approved pharmaceutical drugs; established synthesis protocols exist
• Biocompatibility: cleavage products (thiols) are physiological molecules
• Controlled release: cleavage rate can be modulated through chemical modifications

5. SYNERGISTIC MECHANISMS OF ANTIBACTERIAL ACTION

5.1. Three Distinct Targets

The proposed hybrid system attacks the bacterial cell from three different sides:

1. Moxifloxacin (DNA synthesis inhibition):
   • Penetrates inside the cell after linker cleavage
   • Blocks DNA gyrase and topoisomerase IV
   • Halts DNA replication and cell division
2. THC (membrane disruption):
   • Incorporates into the lipid bilayer of the cell membrane
   • Disrupts its integrity and permeability
   • Reduces ATP levels (energy starvation)
   • Increases reactive oxygen species levels
3. Silver nanoparticles (oxidative stress):
   • Generate reactive oxygen species
   • Interact with protein thiol groups, inactivating them
   • Damage DNA
   • Enhance membrane permeability

5.2. Overcoming THC Ineffectiveness Against Gram-Negative Bacteria

A key advantage of the proposed system is addressing the problem of THC ineffectiveness against gram-negative bacteria. Studies show that if THC is administered together with another agent that “pokes holes” in the outer membrane, THC can penetrate and kill the gram-negative bacterium .

In the proposed system, silver nanoparticles act as such a “battering ram”:

1. AgNPs damage the outer membrane of gram-negative bacteria
2. THC, bound to AgNPs via β-CD, gains access to the inner membrane
3. Moxifloxacin, which itself penetrates well through the outer membrane, enhances the overall effect

5.3. Impact on Biofilms

Biofilms are a primary cause of chronic infections and treatment resistance. THC has a proven ability to disrupt MRSA biofilms. Silver nanoparticles also penetrate biofilms and destroy their structure. The combination of these two agents could ensure effective elimination of bacterial biofilm forms.

6. SYNTHESIS STRATEGY

Based on the analysis of literature data , the following synthesis strategy is proposed:

6.1. Stage 1: Synthesis of β-CD-AgNPs

1. Preparation of a saturated β-cyclodextrin solution (0.2% w/v)
2. Addition of AgNO₃ solution (0.003 mol/L) as a source of Ag⁺ ions
3. Addition of freshly prepared NaBH₄ (0.05 mol/L) as a reducing agent
4. Stirring for 30 minutes until a yellow-brown color appears
5. Centrifugation at 4000 rpm to isolate the nanoparticles

6.2. Stage 2: Formation of the β-CD-AgNPs Complex with THC

1. Addition of THC solution to the β-CD-AgNPs suspension
2. Incubation with stirring to form the inclusion complex
3. Monitoring complex formation by ¹H NMR (appearance of characteristic THC signals) and DSC (disappearance of the THC endothermic peak)

6.3. Stage 3: Synthesis of the Moxifloxacin-Disulfide Linker Conjugate

1. Activation of the moxifloxacin carboxyl group
2. Attachment of the disulfide linker via an amide bond
3. Product purification by chromatography
4. Structure confirmation by NMR and mass spectrometry

6.4. Stage 4: Conjugation with the β-CD-AgNPs-THC Complex

1. Attachment of the moxifloxacin-linker conjugate to THC
2. Purification of the final product
3. Characterization of the complex by physicochemical methods

7. PROSPECTS AND CHALLENGES

7.1. Expected Advantages

• Triple mechanism of action: prevents rapid resistance formation
• Broad spectrum: efficacy against gram-positive, gram-negative bacteria, and anaerobes
• Activity against biofilms: ability to destroy bacterial biofilm forms
• Intracellular delivery: controlled moxifloxacin release inside the cell

7.2. Potential Challenges

• Toxicity: need for thorough assessment of the triple combination’s toxicity in cell cultures and in vivo
• Stability: ensuring the complex’s stability under physiological conditions
• Pharmacokinetics: studying the distribution, metabolism, and excretion of the hybrid system
• Legal aspects: work with THC may be restricted by legislation

8. CONCLUSIONS

1. The design of the hybrid nanocomposite Moxifloxacin—S—S—THC ⊆ β-Cyclodextrin—AgNP for overcoming antibiotic resistance is proposed.
2. The selection of moxifloxacin as the optimal fluoroquinolone is justified due to its broadest spectrum of action, high lipophilicity, and convenient structure for linker attachment.
3. It is shown that β-cyclodextrin performs a dual function: stabilizing silver nanoparticles and forming an inclusion complex with the hydrophobic THC molecule, solving its solubility problem.
4. The use of a disulfide linker is proposed to ensure the intracellular release of moxifloxacin under the action of glutathione.
5. The synergistic mechanisms of antibacterial action of the three components are analyzed, including DNA synthesis inhibition, cell membrane disruption, and induction of oxidative stress.
6. A synthesis strategy for the hybrid nanocomposite is developed based on current literature data.
7. The prospects and potential challenges for further research are identified.

9. ACKNOWLEDGMENTS

The author expresses sincere gratitude to the scientific supervision and colleagues for fruitful discussions and valuable recommendations during the preparation of this work.

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