Doxycycline release and antibacterial activity from PMMA/PEO electrospun fiber mats

Abstract Objective: To investigate the use of polymethyl methacrylate (PMMA) electrospun fiber mats containing different amounts of polyethylene oxide (PEO) as a doxycycline delivery system and to test antibacterial activity against an oral pathogen. Methodology: PMMA powders or PEO (mol wt 200 Kd) (10,20,30% w/w/) were dissolved in N, N-dimethylformamide (DMF) to obtain a final polymer concentration of 15% in DMF (w/v). 2% Doxycycline monohydrate was added to the solutions and submitted to vortex mixing. The solution was transferred to a plastic syringe and fit into a nanofiber electrospinning unit. The parameters applied were: voltage at 17.2 kV; distance of 20 cm between the needle tip and the collector plate; target speed at 2 m/min; and transverse speed at 1cm/min. Syringe pump speed was 0.15 mm/min. The drug release analysis was performed by removing aliquots of the drug-containing solution (in PBS) at specific periods. Doxycycline release was quantified using RP-HPLC. Fiber mats from all groups had their antibacterial action tested against S. mutans based on inhibition halos formed around the specimens. The experiments were performed in triplicate. Gravimetric analysis at specific periods was performed to determine any polymer loss. Morphological characterization of the electrospun fibers was completed under an optical microscope followed by SEM analysis. Results: The addition of PEO to the PMMA fibers did not affect the appearance and diameter of fibers. However, increasing the %PEO caused higher doxycycline release in the first 24 h. Fibers containing 30% PEO showed statistically significant higher release when compared with the other groups. Doxycycline released from the fibers containing 20% or 30% of PEO showed effective against S. mutans. Conclusion: The incorporation of PEO at 20% and 30% into PMMA fiber mat resulted in effective drug release systems, with detected antibacterial activity against S. mutans.


Introduction
Electrospinning is an electrostatic fiber fabrication technique. It has been subject of studies due to its versatility and potential for applications in diverse fields, including tissue engineering, biosensors, filtration, wound dressings, drug delivery, and enzyme immobilization. 1,2 The sub-micron range spun fibers produced offer various advantages, such as high surface area to volume ratio, tunable porosity (when spun into a mat) and ability to manipulate nanofiber composition to get the properties desired. 1 Recently, nanofibers have been used in different Dentistry fields, including composite reinforcement, 3,4 periodontal regeneration, 5 and implant surface treatments. 6 They are also used as scaffolds for dental tissue and bone regeneration. 7 However, the greatest potential of these fibers in Dentistry is the use of mats as drug delivery systems. The ability to produce drugloaded polymeric fiber meshes or mats may offer an enhanced drug release profile compared with other polymeric drug release implants. As in many other fields, this mechanism can be convenient in Dentistry in multiple contexts. 8 Among several polymers able to form ultrafine fibers in the electrospinning process, polymethyl methacrylate (PMMA) is a reliable biocompatible option, commonly used in restorative Dentistry. 9 Electrospinning a blend of PMMA and polyethylene oxide (PEO) may result in biocompatible and toughness-enhanced ultra-thin fiber mats very useful for biomedical applications. 10 PEO is frequently used as a co-spinning component in electrospinning solutions to offset problems related to continuous fiber formation without structural imperfections 11 or even as a plasticizer to allow fiber formation when blended with different polymers, such as cellulose acetate. 12 Doxycycline is an antimicrobial drug proved effective against oral bacteria, especially for periodontal disease. 13 It presents anti-collagenase activity 14 as additional advantage, which could be beneficial when treating sites sometimes compromised by undue metalloproteinase (MMP) activity such as at bonded interfaces with dentin. 15 The described polymers formed into fibers loaded with antibacterial and anti-enzymatic drugs, as doxycycline, are expected to be an important controlled drug delivery tool in Dentistry. One considered that the addition of PEO would not affect the homogeneity or diameter or PMMA fibers formed into mats, but that the inclusion of a water soluble polymer such as PEO might increase drug release rates and antibacterial activity of the mats. Thus, the main objectives of this study were to produce PMMA fiber mats using two room temperature before electrospinning. The solution was transferred to a 10 mL plastic syringe and fit into a nanofiber electrospinning unit (NEU -Kato Tech, Japan). Electrospinning manufacture of nanofibers depends on many parameters, including the polymer molecular weight and concentration, flow rate, needle/ collector distance, target speed, and applied voltage.
Every polymer-drug combination needs to be spun under different conditions to achieve an optimal fiber production. This is usually optimized visually whereby the operator begins with intermediate values for these factors and sees how the polymer spins. In this study, a 15% polymer solution was associated with non-sputtering spinning and uniform fiber production, which could be seen to "draw" off the needle bead and a white mat appeared on the collection drum.  with 30% PEO loaded films were statistically significant at all periods up to 48 hours (also, 20% PEO was higher than 10% PEO).

Gravimetric analysis
All fiber mats lost weight over time when placed in water ( Figure 2). For 10% PEO loaded films, weight loss occurred in less than one hour with no further weight loss after that. For 20% PEO loaded mats, weight loss increased over 8 hours and then plateaued with no further loss. Similarly, for 30% loading, weight loss was complete at 8 hours. As PMMA is waterinsoluble, the assumption was that all weight loss occurred due to PEO release. For 10% PEO loaded fiber mats, less than 20% of encapsulated PEO was released from the fibers. For 20% and 30% loadings, almost 50% of the PEO was released after 8 hours.

Antibacterial analysis
The initial concentration estimated for S. mutans was 5.0×10 8 CFU/mL. Both 20% and 30% PEO loaded doxycycline fibers inhibited the growth of S. mutans as seen in Figure 3. These inhibition zones were similar to those resulting from non-encapsulated doxycycline in solution from filter paper.   PMMA is a well-known polymer, commonly called acrylic, used for temporary crowns, complete or partial dentures, orthodontic appliances, and personalized trays for impressions among its dental applications.
Its qualities of biocompatibility, reliability, relative ease of manipulation, and low toxicity were soon seized upon and incorporated by many different medical specialties. 24,25 PMMA is hydrophobic and dimensionally stable. Therefore, it is a versatile polymer in drug delivery systems and has been used in various areas of the biomedical field: it has been proven to be an efficient antimicrobial agent delivery device, preventing in vivo Staphylococcus aureus biofilm formation. 26 On the other hand, as this is a hydrophobic polymer, it does not swell at all in water.
Other biocompatible polymers, such as poly (lacticco-glycolic) (PLGA), are more commonly used in electrospinning methods for drug delivery because they swell in water and release drugs over a long period, until degradation. 27,28 . However, polymers that distort in water are not suitable for most dental applications requiring mechanical stability.
In this study, we showed little change in fiber diameter after the immersion of mats in water, making even 30% PEO loaded mats suitable for producing dimensionally stable fibers. Morphological characteristics of the fibers, such as diameter, may have a significant impact on the drug release. 33 In this study, it was initially challenging to produce uniform, beadless fiber mats from PMMA.
A pilot study revealed that the best formulation combined two different molecular weights of PMMA (75K and 996K) in pure DMF. The electrospun fibers formed were uniform, well distributed, and beadless.
The fiber mats obtained were easy to handle. No statistically significant difference was found in the mean diameter among the groups. Although the fibers were relatively large in diameter (ca.>1000 nm) considering that nano level fibers can be produced, they were uniform among the groups and allowed us to interpret our findings based exclusively on the PEO concentration variable. Reproducibility and uniformity are more important than reduced diameter. 9 Multiple alternative strategies to the use of antibiotics have been applied to enhance antimicrobial activity of polymeric fiber meshes, including the incorporation of antimicrobial metals, semi-metals, or graphene compounds. 34,35 . In this study, doxycycline was the drug selected for the experiment because it is a broad-spectrum antibiotic drug, which has been shown to be active against both gram-positive and gram-negative organisms. Streptococcus mutans was selected due to its clinical relevance on metabolic-induced dental tissue loss. Doxycycline has been successfully used to control the progression of periodontal disease by systemic and localized delivery at antimicrobial levels. The drug has also been encapsulated within nanotube-modified dentin adhesive, presenting sub-antimicrobial concentrations and ability to inhibit MMPs. 13,[36][37][38][39] In future studies using PMMA/PEO fiber, drug release profiles for specific dental applications need to be better quantified. However, the concept

Conclusion
Based on the experiments performed, one can conclude that consistent production of PMMA/ PEO eletrospun fiber mats with adequate handling properties to be used in dental applications was possible. PEO inclusion influenced positively the doxycycline release profile from PMMA fiber mats, but did not alter the morphological characteristics of the fibers. PMMA fiber mats containing 20% and 30% PEO and 2% doxycycline presented antibacterial activity against S. mutans.