Linear copolymers based on cholinium functionalized with antibiotic anions for single– and dual–drug delivery systems

Linear copolymers based on cholinium functionalized with antibiotic anions for single– and dual–drug delivery systems | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Linear copolymers based on cholinium functionalized with antibiotic anions for single– and dual–drug delivery systems Shadi Keihankhadiv, Dorota Neugebauer This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4580822/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The novel single and dual drug delivery systems (DDS) were designed by controlled polymerization of pharmaceutically functionalized choline-based monomers, i.e. [2-(methacryloyloxy)ethyl]trimethylammonium with counterions of cloxacillin (TMAMA/CLX), and ampicillin (TMAMA/AMP), providing the properties of antibiotics. This strategy was convenient to attain the well-defined linear copolymers with 38–93 mol. % of TMAMA contents, which were regulated by the initial ratio of TMAMA to methyl methacrylate comonomer. The compositions of polymers were controlled by the total monomer conversion (40–75%) resulting in a variable degree of polymerization (DP n = 160–300) and contents of pharmaceutical anions (CLX¯ 51–80% and AMP¯ 78–87%). In aqueous solution, particles of the polymer achieved nanoscale sizes, measuring between 274–380 nm for CLX¯ systems and 288–348 nm for CLX¯/AMP¯ systems. In vitro drug release, which was driven by the exchange reaction of the pharmaceutical to phosphate anions in PBS, imitating a physiological fluid, occurred in the range of 58–76% of CLX¯ (10.5–13.6 µg/mL) in the single systems, and 91–100% of CLX¯ (12.9–15.1 µg/mL) and 97–100% of AMP¯ (21.1–23.3 µg/mL) in the dual systems. In relation to the conventional systems delivering both antibiotics without polymer carrier, the studied choline-based polymer DDS, demonstrating effective content of drug(s) and their (co)release from the polymer carriers, seems to be a great alternative solution. Physical sciences/Chemistry/Medicinal chemistry Physical sciences/Chemistry/Polymer chemistry Physical sciences/Chemistry/Synthesis Physical sciences/Chemistry Physical sciences/Materials science linear polymers ampicillin cloxacillin ionic liquid choline drug delivery system Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Choline, identified as 2-hydroxyethyl trimethylammonium chloride, is naturally synthesized by the human liver and is present in phospholipids like phosphatidylcholine or lecithin. This organic salt represents ionic liquids (ILs) [ 1 ], which possess chemical stability, ability to enhance solubility, and modifiability by ion exchange for adjustment of physical and chemical properties [ 2 , 3 , 4 ]. Their biological attributes include enhancing skin penetration [ 5 ], acting as antibacterial properties [ 6 ], functioning as stabilizers [ 7 , 8 ], cytotoxic and local anesthetic properties, anti-fungal and anti-acne activities, and antibiotic actions [ 9 , 10 , 3 ]. Furthermore, the versatility of ILs extends to their ability to accommodate a wide range of pharmaceutical substances, including antiviral and antimicrobial agents, antioxidants, anticoagulants, nonsteroidal anti-inflammatory drugs, anticancer drugs, and others [ 11 , 12 , 13 , 14 , 15 , 16 , 3 ]. The widely employed cholinium cation offers biodegradability, water-solubility, and low cost for various applications [ 17 , 18 , 19 ]. Its properties have been investigated in combination with various bioactive compounds, including phenytoin [ 20 ], ampicillin (AMP) [ 21 ], nalidixic acid, niflumic acid, p -aminosalicylic acid, pyrazinoic acid, and picolinic acid [ 18 ]. These systems have demonstrated enhanced solubility for the active pharmaceutics, elevating their capability to permeate the cell membrane [ 17 ]. In recent decades, the synthesis of precisely designed polymers with the desired architecture, composition, chain homogeneity, site-specific functionality [ 22 , 23 , 24 , 25 ], physicochemical and biochemical attributes (e.g. mechanical strength, softness, self-healing, processability, tissue adhesiveness, bioactivity, and biodegradation) [ 26 , 27 ] appeared to be a powerful material enabling the development of versatile nanostructures applicable in biology and medicine [ 28 ]. The tailored-made polymers synthesized by the controlled polymerization methods had influenced on significant progress in drug delivery systems (DDS) offering linear and branched polymer carriers with bio-therapeutics functions [ 29 , 30 , 31 ]. Both, drug conjugated or encapsulated by polymers have been intensively studied to address challenges related to the drug's hydrophilicity [ 32 , 33 ]. Furthermore, the delivery of more than one bioactive compound has been tested to enhance the main drug's activity [ 34 , 35 , 36 ]. The commercial choline ester derivative, [2-(methacryloyloxy)ethyl]trimethyl-ammonium chloride, referred to methacroylcholine (TMAMA/Cl), functions as a choline-based ionic liquid, which serves as the monomer for obtaining the polymerized ionic liquid (PIL) [ 37 ]. This PIL has been reported as delivering pharmaceutical anions through the anion exchange in the polymer matrix[ 38 , 39 , 40 ] or encapsulating various bioactive compounds [ 40 , 41 , 42 , 43 , 44 , 45 , 46 ] to create a pharmaceutically active polymeric systems. The pharmaceutically active choline-based PILs have been also designed by direct polymerization of pharmaceutically functionalized choline monomers with salicylate [ 37 , 47 , 48 ], p -aminosalicylate [ 49 , 50 ], fusidate [ 51 ], and cloxacillin (CLX)[ 51 ] counterions. In this research, we investigated the well-defined linear copolymers based on the biofunctionalized choline ionic liquids with ionically conjugated AMP and CLX as the pharmaceutical anions. Depending on the strategy employed, the linear copolymers were designed either as single DDS carrying CLX¯ or dual DDS with CLX¯ and AMP¯ (Fig. 1 ), where the drug anion is linked via an ionic bond to a polymer matrix. Both drugs are antibiotics deriving from semi-synthetic penicillin, which demonstrates antimicrobial efficacy from the existence of a beta-lactam ring [ 52 , 53 ]. Because of effectiveness against both gram-positive and some gram-negative microorganisms [ 54 ] they are employed for the treatment of bacterial infections affecting the ear, nose, throat, bones, lungs, as well as post-operative wound infections [ 55 ]. The commercially available combination of AMP and CLX is marketed under various brand names, such as Ampiclox, Apen, Cloxam, and Megamox, in the form of capsules or oral suspensions. Therefore, both pharmaceuticals are well-suited for utilization in either individual treatment or combined therapies. The obtained polymer systems were characterized to show their effectiveness in drug delivery through the evaluation of drug content and (co)release during an in vitro study. The advantage of this novel DDSs is featured by the selected antibiotics carried by the ionic polymers, where pharmaceutical anions are released by physiological solution by exchange with phosphate anions. 2. Materials and Methods Methyl methacrylate (MMA, Alfa Aesar, Warsaw, Poland), tetrahydrofuran (THF, Sigma Aldrich, Poznan, Poland) and methanol (MeOH, Chempur, Piekary Śląskie, Poland) were dried using molecular sieves (type 4A, bulk density 640–670 kg/m 3 , Chempur, Piekary Śląskie, Poland) under argon. [2-(Methacryloyloxy)ethyl]trimethylammonium chloride (TMAMA/Cl, 80% aq. solution, Sigma-Aldrich, Poznan, Poland) was concentrated under vacuum until it achieved solid product. Copper (I) bromide (CuBr, Fluka, Steinheim, Germany) was purified by stirring in glacial acetic acid, followed by filtration, and washing with ethanol and diethyl ether, and subsequent vacuum drying. Deionized water was obtained using the Hydrolab HLP Uv5 equipment (Straszyn, Poland). Ethyl 2-bromoisobutyrate (EBiB), phosphate-buffered saline (PBS, pH 7.4), N , N , N ′, N ″, N ″-pentamethyldiethylenetriamine (PMDETA) and ampicillin sodium salt (NaAMP) from Sigma Aldrich (Poznan, Poland), cloxacillin sodium monohydrate (NaCLX) and deuterated dimethyl sulfoxide (DMSO–d6) from Alfa Aesar, as well as N,N -dimethylforamide (DMF, POCH, Gliwice, Poland), phosphate buffer solution (PBS, Sigma Aldrich, Poznan, Poland) and diethyl ether (Chempur, Piekary Śląskie, Poland), were used without prior purification. 2.1. Preparation of pharmaceutically functionalized choline ionic liquids by ion exchange The vacuum dried TMAMA/Cl (2.14 mmol, 0.445 g) was dissolved in 2.2 mL of MeOH (forming solution 1). Next, NaCLX (2.14 mmol, 1.02 g) was dissolved in 5.1 mL of MeOH (TMAMA/Cl: MeOH = 1:5 w/v) and added dropwise to solution 1 while the mixture stirred continuously during drug addition. Then, the mixture was stirred for 3 hours constantly in a dark place at room temperature during the ion exchange reaction. After NaCl salt precipitation, the solution was filtered and washed twice with 1 mL MeOH to remove any salt. To accelerate MeOH evaporation, the filtrated solution containing TMAMA/CLX was left on a ventilator at room temperature for 30 minutes. It was further dried under vacuum until it solidified into a powder product. Yield: 1.26 g. 1 H NMR (DMSO–d 6 ,300 MHz, δ, ppm): 7.4–7.7 (4H, aromatic ring of CLX), 5.75 and 6.15 (2H, =CH 2 in vinyl group), 5.25–5.45 (2H, -CH-N- and -CH-S- in β-lactam ring of CLX), 4.54 (2H, -CH 2 -O-), 3.82 (1H, -CH-N- in thiazolidine ring of CLX), 3.73 (2H, -CH 2 -N + -), 3.14 (9H, -N + (CH 3 ) 3 ), 2.65 (3H, -CH 3 at isoxazole ring in CLX), 1.92 (3H, -CH 3 ), 1.46 (6H, -C(CH 3 ) 2 in thiazolidine ring of CLX). The synthesis of analogical TMAMA/AMP followed the previously described method, utilizing equimolar ratios of TMAMA/Cl (3.9 mmol, 0.8 g) and AMPNa (3.9 mmol, 1.43 g) in MeOH (4 mL and 7.2 mL, respectively). However, the ion exchange process was completed within 20 hours. Yield: 2.04 g. 1 H–NMR (DMSO-d 6 ,300 MHz, δ, ppm): 7.1–7.5 (5H, -CH in aromatic ring in AMP), 5.75 and 6.15 (2H, -CH 2 in vinyl group), 5.25–5.5 (2H, -CH-N- and -CH-S- in β-lactam ring of AMP), 4.54 (2H, -CH 2 -O-), 4.46 (1H, -CH-NH 2 in AMP), 3.8-4.0 (1H, -CH-N- in thiazolidine ring of AMP), 3.75 (2H, -CH 2 -N + -), 3.16 and 3.12 (9H, -N + (CH 3 ) 3 ), 1.92 (3H, -CH 3 ), 1.57 and 1.46 (6H, -C(CH 3 ) 2 in thiazolidine ring of AMP). 2.3. Synthesis of copolymer conjugates 2.3.1. Ionic linear copolymers containing CLX anion, P(TMAMA/CLX – co – MMA) (Example for IA) TMAMA/CLX (0.656 mmol, 0.41 g) was dissolved in 0.41 mL of MeOH in a Schlenk flask. Then, MMA (1.968 mmol, 0.21 mL), THF (0.14 mL), and PMDETA (0.007 mmol, 0.001 mL) were added to the flask. The mixture underwent homogenization and degassing by two freeze–pump–thaw cycles. Then, EBiB (0.007 mmol, 0.001 mL) was introduced as an initiator, and the initial sample was taken. Another freeze–pump–thaw cycle was performed. Following this, CuBr catalyst (0.007 mmol, 0.001 g) was quickly added to the mixture, and the reaction flask was immersed in an oil bath at 40°C. After 2 hours, when a noticeable increase in the mixture's viscosity was observed, the reaction was stopped by exposing it to air. Subsequently, the resulting product was dissolved in MeOH (1 mL) and precipitated twice in a chloroform–diethyl ether mixture (1:1) to eliminate the catalyst from the polymer. Solvents were discarded using a syringe, and the remaining solvents were evaporated under ventilator at room temperature for 15 minutes. Finally, the polymer was dried under the vacuum. Yield: 0.24 g. 1 H–NMR (DMSO–d 6 , 300 MHz, δ, ppm): 7.4–7.7 (4H, aromatic ring of CLX), 5.3-5,5 (2H, -CH-N- and -CH-S- in β-lactam ring of CLX), 4.1–4.3 (2H, -CH 2− O-), 3.82 (1H, -CH-N- in thiazolidine ring of CLX), 3.60–3.75 (2H, -CH 2 -N + -), 3.5–3.6 (3H, -O-CH 3 ), 3.02–3.30 (9H, -N + (CH 3 ) 3 ), 2.65 (3H, -CH 3 at isoxazole ring in CLX), 1.75 (2H, -CH 2 -C-), 1.36–1.46 (6H, -C(CH 3 ) 2 in thiazolidine ring of CLX), 0.54–1.2 (3H, -CH 3 ). 2.3.2. Dual bioactive ionic linear copolymers containing CLX¯/AMP¯, P(TMAMA/CLX – co – MMA – co – TMAMA/AMP) (Example for IIA) TMAMA/CLX (0.557 mmol, 0.35 g) and TMAMA/AMP (0.557 mmol, 0.29 g) were dissolved in 0.64 mL of MeOH in a Schlenk flask. Then, MMA (3.342 mmol, 0.36 mL), THF (0.21 mL), and PMDETA (0.011 mmol, 0.002 mL) were added to the flask. Further steps were provided like for synthesis and purification of P(TMAMA/CLX– co –MMA) described in section 2.3.1 using proper amounts of EBiB (0.011 mmol, 0.002 mL) and CuBr (0.011 mmol, 0.002 g). Yield: 0.73 g. 1 H–NMR (DMSO–d 6 ,300 MHz, δ, ppm): 7.1–7.7 (9H, -CH in aromatic rings of AMP and CLX), 5.25–5.4 (2H, -CH-NH- and -CH-S- in β-lactam ring of CLX and AMP), 4.43 (1H, -CH-NH 2 in AMP), 4.1–4.3 (2H, -CH 2− O-), 3.75–3.87 (2H, -CH-N- in thiazolidine ring of CLX and AMP), 3.65–3.75 (2H, -CH 2 -N + -), 3.4–3.65 (3H, -O-CH 3 ), 3.05–3.25 (9H, -N + (CH 3 ) 3 ), 2.65 (3H, -CH 3 at isoxazole ring in CLX), 1.75 (2H, -CH 2 -C), 1.4–1.7 (12H, -C(CH 3 ) 2 in thiazolidine ring of CLX and AMP), 0.6–1.2 (3H, -CH 3 ). 2.4. Release studies of copolymer conjugates containing CLX¯ and AMP¯ Copolymer conjugates containing drug anions (1 mg) were dissolved in 1 mL of PBS (pH 7.4) to achieve a 1 mg/mL solution. The solution was then transferred into a dialysis cellulose membrane bag with a molecular weight cutoff (MWCO) of 3.5 kDa. Then, this membrane bag was placed inside a glass vial filled with 44 mL of PBS and stirred at 37°C during a 74–hour dialysis process. The release progress was monitored by assessing changes in drug concentration in the external PBS solution surrounding the dialysis bag. At different time intervals, 0.5 mL samples of the solution containing the released drug were collected, and 0.5 mL of MeOH was added to the cuvette. These samples were subsequently analyzed using UV-Vis spectrophotometer to quantify the released drug amount by measuring the absorbance at a wavelength of λ = 206 nm for AMP¯ and λ = 203 nm for CLX¯. Each reported result represents the average of three measurements. 2.5. Characterization methods Proton nuclear magnetic resonance ( 1 H–NMR) spectra were acquired using a UNITY/NOVA spectrometer (Varian, Mulgrave, Victoria, Australia) operating at frequency of 300 MHz. The measurements were conducted on samples dissolved in DMSO–d 6 with tetramethylsilane used as the internal standard. For size exclusion chromatography (SEC), an Ultimate 3000 chromatograph (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a differential refractometer detector RefractoMax 521 was employed. The polymer samples prepared in DMF containing 10 mM LiBr at 40°C, were passed through a precolumn TSKgel Guardian SuperMP(HZ)-H (4.6 mm × 2 cm, with a particle size of 6 µm), followed by two columns of TSKgel SuperMilipore HZ-H (4.6 mm × 15 cm, with a particle size of 6 µm). The flow rate was maintained at 0.45 mL/min. The average molecular weight (M n ) and dispersity index (Ð) were determined on the base of poly(ethylene oxide)/poly(ethylene glycol) (PEO) standards with molecular weights ranging from 982 to 969,000 g/mol. The drug content (DC) and the amount of released drug (ARD) were analyzed by ultraviolet-visible light spectroscopy (UV-Vis) employing a spectrometer Evolution 300 (Thermo Fisher Scientific, Waltham, MA, USA). Sample of the polymer dissolved in PBS and MeOH (1:1) at a concentration of 0.05 mg/mL were placed in a quartz cuvette and measured at a maximum wavelength of λ = 206 nm for AMP¯ and λ = 203 nm for CLX¯. A calibration curve was established for drug concentrations ranging from 0.1 mg/mL to 0.006 mg/mL in the mix of PBS and MeOH (1:1). Dynamic light scattering (DLS) was performed using Nanotrac Flex with laser particle size analyzer Mictrac MRB's (Microtrac Retsch GmbH, Haan, Germany; Dimensions LS software 1.1.0.), equipped with an external “dip-in” probe with 180 0 backscattering. Polymer samples were dissolved in deionized water at a concentration of 1.0 mg/mL, and measurements were repeated for each sample at least three times to analyze the hydrodynamic diameters (Dh) of polymer nanoparticles and their polydispersity index (PDI). 3. Result and Discussion 3.1. Monomeric ionic liquid functionalized by pharmaceutical anions The proposed strategy to pharmaceutically functionalized copolymers included the modification of the water-soluble ionic liquid, that is [2-(methacryloyloxy)ethyl]-trimethylammonium chloride (TMAMA/Cl) with polymerizable methacrylate group and Cl¯, where the latter one was exchanged by CLX¯ and AMP¯ becoming from their sodium salts (NaCLX and NaAMP). This exchange reaction resulted in a monomers carrying a pharmaceutical anion, namely [2-(methacryloyloxy)ethyl]trimethylammonium cloxacillin (TMAMA/CLX) and ampicillin (TMAMA/AMP) as depicted in Scheme 1 and Fig. 1 . The monomer structures were confirmed by the 1 H-NMR spectra to evaluate the efficiency of ion exchange by assigning characteristic proton signals to TMAMA, especially 9 protons in the trimethylammonium cation TMAMA units (D), and the pharmaceutical counterions (Fig. 2 ). In the case of TMAMA/AMP the signal D at 3.16 ppm was still existing after exchange, but a new one appeared at 3.12 ppm, which demonstrated a 47% of exchange efficiency (Fig. 2 b), whereas for TMAMA/CLX it was slightly shifted to 3.14 ppm indicating 100% of the anion exchange (Fig. 2 c). 3.2. Linear polymers containing CLX¯ as single drug delivery systems The above-described ionic monomer TMAMA/CLX, with a choline species and pharmaceutically active counterion was copolymerized with the non-ionic monomer MMA in molar ratios of 25/75, 50/50, and 75/25 to synthesize the well-defined linear copolymers, denoted as P(TMAMA/CLX– co –MMA)s (IA–IC) as the single drug systems carrying different ionic content of TMAMA/CLX. For this purpose, the ATRP reactions were initiated by EBiB as a monofunctional initiator, catalyzed by CuBr/PMDETA complex, dissolved in MeOH/THF solvent mixture, and conducted at 40°C (Fig. 1 ). The conversion of the monomer to polymer and polymer structure were confirmed by 1 H–NMR spectroscopy (Fig. 3 ). The calculation of total conversions of both monomers (X) was relied on a broad signal deriving from protons of the methyl group in the formed polymer backbone (B, 0.54–1.2 ppm) in relation to the signals from a proton in the vinyl groups of unreacted TMAMA and MMA monomers (identified as M1 and M2, in the range of 5.25 to 6.25 ppm). Moreover, the proton signal in the vinyl group of the unreacted monomer M1 at 6.09 ppm and the proton signal associated with the trimethylammonium group present in both the monomer and resulting polymer (referred to F at 3.02–3.30 ppm) were utilized to determine the TMAMA conversion (X M1 ). Further, the conversion values were utilized to calculate other parameters, such as polymerization degree (DP), the ionic fraction content in the copolymer (F M1 ), and the average molecular weight of the copolymer (M n ), which are presented in Table 1 . Table 1 Characteristics of linear copolymer P(TMAMA/CLX– co –MMA)’s synthesized by ATRP. No. f M1 /f M2 (mol%) Time (h) X M1 a (%) X a (%) DP M1 a DP n a F M1 a (mol) M n a (g/mol) M n b (g/mol) Ð b IA 25/75 2 87 40 87 160 0.54 59900 10700 1.24 IB 50/50 90 60 179 241 0.74 115100 - - IC 75/25 92 75 277 299 0.93 170200 - - Where: M1 = TMAMA/CLX, M2 = MMA; conditions: [TMAMA/CLX + MMA] 0 : [EBiB] 0 : [CuBr] 0 : [PMDETA] 0 = 400:1:1:1; MeOH:TMAMA/CLX = 1:1 υ/ωt, MeOH:THF = 3:1 υ/υ, 40 o C; f M1 , f M2 – initial content of monomer in the reaction mixture; X M1 a –TMAMA conversion; X - total monomer conversion; DP M1 - polymerization degree of ionic monomers; DP n - total polymerization degree; F M1 - content of ionic fraction in the polymer; M n - average molecular weight; Đ – dispersity index. a determined by 1 H-NMR (DMSO-d6), b determined by SEC (DMF, PEO calibration). Linear copolymers were obtained within a short time of 2 h by total monomer conversions ranging from 40–75%, while the conversion of ionic monomer remained similar (87–92%). The observed tendency of the monomer conversion increases with the initial content of TMAMA monomer shows that the latter factor can be used to regulate the length of polymer chain (DP n =160–299) and the amount of ionic units (DP M1 =87–277). The compositions of the resulting copolymers IA–IC revealed a higher content of the ionic fraction than it was anticipated by the initial content of the ionic monomer (f M1 /F M1 : 25/54 (IA); 50/74 (IB); 75/93 (IC)). This phenomenon indicates higher reactivity of the ionic monomer (TMAMA/CLX), which is favored over MMA under polar conditions, probably leading to the formation of copolymers with gradient structures. 3.3. Linear polymers containing CLX¯/AMP¯ as dual drug delivery systems The dual drug anions carried by polymer conjugates resulted by the terpolymerization of two pharmaceutically functionalized choline monomers, i.e. both TMAMA/CLX and TMAMA/AMP with MMA in the ratio equal to 12.5:12.5:75, 25:25:50, 37.5:37.5:25 via ATRP under conditions similar to those above described for the synthesis of single drug copolymer conjugates (Fig. 1 ). The P(TMAMA/CLX– co –MMA– co –TMAMA/AMP)s (IIA–IIC, Table 2 ) were synthesized to be capable of transporting two drugs concurrently within the system. Figure 4 shows the assessment of monomers converted into the polymers through 1 H–NMR analysis, employing analogous signals as previously described for series IA–IC. Table 2 Characteristics of linear terpolymers P(TMAMA/CLX– co –MMA– co –TMAMA/AMP)’s synthesized by ATRP. No. f M1 /f M2 (mol%) Time (h) X M1 a (%) X a (%) DP M1 a DP n a F M1 a (mol) M n a (g/mol) M n b (g/mol) Ð b IIA 25/75 2 91 60 91 239 0.38 55500 16200 1.58 IIB 50/50 3.5 90 58 181 231 0.78 85400 - - IIC 75/25 3 92 75 275 300 0.92 124800 - - Where: M1 = TMAMA/CLX + TMAMA/AMP, M2 = MMA; conditions: [(TMAMA/CLX + TMAMA/AMP) + MMA] 0 : [EBiB] 0 : [CuBr] 0 : [PMDETA] 0 = 400:1:1:1; MeOH:TMAMA = 1:1 υ/ωt, MeOH:THF = 3:1 υ/υ, 40 o C; f M1 , f M2 – initial content of comonomers in the reaction mixture; X M1 a – conversion of both TMAMA monomers; X - total conversion; DP M1 - polymerization degree of ionic monomers; DP n - total polymerization degree; F M1 - content of ionic fraction in the polymer; M n - average molecular weight of copolymer; Đ – dispersity index. a determined by 1 H-NMR (DMSO-d6), b determined by SEC (DMF, PEO calibration). The presence of the second drug based TMAMA in the polymerization with the lowest initial content of ionic monomers affected the formation of significantly higher chain lengths and similar amounts of incorporated ionic units when comparing IIA with IA (DP n /DP M1 = 239/91 vs 160/87). This observation suggests possible differences in the relative reactivities of ionic comonomers, where TMAMA with AMP counterion appearing to be more reactive than that with CLX anion, which presents higher steric hindrance resulting from its Cl-substituted aromatic ring and extra 5-membered ring. Additionally, due to its structure, CLX seems to be less hydrophilic than AMP, making TMAMA/AMP more favorable under the polymerization conditions used. In relation to MMA, both TMAMAs polymerized faster, as evidenced by the correlation F M1 > f M1 . In the system of three monomer components, MMA was converted at a higher rate than in two monomer system, leading to lower ionic content in polymer IIA compared to IA (38% vs 54%). However, a higher initial concentration of TMAMAs (f M1 = 50 and 75%) provided similar total monomer conversions, as well as DP n and DP M1 values, to analogous systems in series I, but it required longer time of polymerization as convenient for incorporation of slower TMAMA/CLX. Generally, knowing these relations the compositions of the resulting polymers IIA–IIC can be assumed by the initial proportions of TMAMA/MMA (f M1 /F M1 : 25/38 (IIA); 50/78 (IIB); 75/92 (IIC)). The SEC data were obtained for copolymers with ionic fraction below 60%, which were well-soluble in DMF. verifying the well-controlled polymerization. The molecular weight distribution for the dual drug polymer IIA was larger than for the single drug IA (Đ=1.58 vs. 1.24), which suggests that the occurrence of side reactions was promoted in the system containing two types of TMAMA counterions with different natures. The drug content (DC), which defines the percentage of pharmaceutical anions included in the polymer chain, was evaluated by UV-Vis spectroscopy. The DC of CLX¯ was ranged in 67–80% in the single (IA–IC) and 51–64% in the dual drug systems (IIA–IIC), where the latter ones also co-delivered 78–87% of AMP¯ (Fig. 5 ). The simultaneous increase of DCs driven by the total polymerization degree, including polymerization degree of TMAMA was observed in the single drug polymers in Fig. 5 a (DC/DP n /DP M1 : 67/160/87 (IA), 73/241/179 (IB), 80/299/277 (IC)). Similarly, the trend of increasing DC correlated with the amount of TMAMA units was also indicated for the dual systems with additional relation between DC of both drugs, that is DC CLX < DC AMP (IIA: 51% CLX¯ vs 78% AMP¯, IIB: 62% CLX¯ vs 83% AMP¯, IIC: 64% CLX¯ vs 87% AMP¯) as demonstrated in Fig. 5 b. In summary, the highest values for CLX¯ were achieved for IC and IIC with the longest polymer chain lengths and the highest ionic contents (DP n ~ 300 and F M1 > 92%), which makes them the most advantageous systems. 3.4. Hydrodynamic diameters of polymer particles Dynamic light scattering (DLS) analysis was conducted to assess the particle size of both types of drug delivery polymer conjugates in an aqueous solution (Table 3 ). The histograms in Fig. 6 depict the distribution of the formed particles exhibiting one fraction. The results for the single drug polymer systems revealed distinct hydrodynamic diameters (Dh) for IA and IC exhibited Dh ≤ 274 nm, whereas IB displayed a larger value of 380 nm. In the dual drug systems (IIA–IIC), despite the differences in polymer structure, not big significant influence was observed on particle sizes, which ranged from 288 nm to 348 nm. Furthermore, the polydispersity index (PDI), which defines the level of heterogeneity of particles as the distribution of their sizes, indicated uniformity of particle sizes (PDI ≤ 0.01). Remarkably narrow size distributions were exhibited by IB and IIC (PDI: 0.006 and 0.008, respectively). Table 3 DLS characteristics of linear polymer particles carrying drug. Type DDS Polymer Dh (nm) PDI Single IA 274 0.06 IB 380 0.006 IC 277 0.01 Dual IIA 288 0.01 IIB 324 0.01 IIC 348 0.008 where: Dh is hydrodynamic diameter and PDI is polydispersity index. 3.4. Drug Release In vitro drug release studies were conducted to monitor the exchange of CLX ¯ or CLX ¯ /AMP ¯ anions in the polymer matrix by phosphate anions in the PBS via the dialysis method under physiological conditions in pH 7.4 at 37°C for 72 hours. Drug release from the polymer samples was detected at specified time intervals, and the percentage amount of released drug (ARD, Fig. 7 a) correlated with the drug concentration in the released medium (CRD, Fig. 7 b) in relation to the DC were determined using UV–Vis spectroscopy. The kinetic profiles for the single drug systems illustrated a release of 34–41% of CLX¯ within 0.5–1 hour, which increased to 58–72% within four hours and continued up to 74 hours, representing relative stability with additional 4–6% of drug release (Fig. 8 a). However, in the case of sample IC the release profile differed from the others, exhibiting a more linear relationship that was initially similar to the system IA, but ultimately reached the highest ARD value. The increase in the amount of drug released from the single systems corresponded well with elevated total polymerization degree and drug content (DP n /DC/ARD: 160/67/58 (IA), 241/73/66 (IB), 299/80/76 (IC)). Combining the effective content of drug and its release, polymer IC appeared to be the most promising for an extended therapeutic effect. In the context of the dual co-delivery systems, a significant initial burst release was observed in the first hour for both drugs, yielding 64–80% for CLX¯ (Fig. 8 b) and 90–98% for AMP¯ (Fig. 8 c). Following the release rate was notably slowed down yielding an additional 12–16% of CLX¯ released within the next 2 hours, and reaching ARD values of 88–100% after 4 hours. For incomplete release, the process was extended up to 74 hours, but slight progress was detected, maintaining a release plateau. In summary, complete release of both drugs was achieved by system IIA within 4 hours, whereas the release from IIB and IIC systems was slightly slower, reaching > 90% of CLX¯ and > 97% of AMP¯. The results for series II suggested that the interactions between CLX¯ and AMP¯ improved the co-release of CLX, although its DC was slightly lower than that in the series I, but the polymers, especially IIA, were able to completely release both drugs in a relatively short time. 4. Conclusions The well-defined linear copolymers, including series I with CLX¯ (single drug DDS) and series II containing both CLX¯ and AMP¯ (dual drug DDS), were synthesized to demonstrate their potential as nanocarriers (274–380 nm (CLX¯) and 288–348 nm (CLX¯/AMP¯)). In both series, the TMAMA monomers functionalized by pharmaceutical anions with antibiotic activities were incorporated into the polymer chains in different contents (38–93 mol%). The various lengths of polymer chains (DP n =160–300) were adjusted by the monomer conversion (40–75%), while the initial proportion of comonomers allowed for regulation of ionic contents (38–93 mol%) in the polymers. The drug content within the polymer matrix, ranging in 67–80% in the CLX systems, and 51–64% of CLX¯ and 78–87% of AMP¯ in the dual drug systems, corresponded to efficient in vitro release of 91–100% of CLX¯ (12.9–15.1 µg/mL) and 97–100% of AMP¯ (21.1–23.3 µg/mL) from the dual drug systems, and 67–80% of CLX¯ in the single drug systems in PBS within 72h. The drug content and release were significantly affected by factors, such as polymer chain length, ionic fraction content in the copolymer, and pharmaceutical anion nature. Generally, linear copolymers demonstrated effectiveness in designing both single– and dual–drug systems with customized release profiles, facilitating drug delivery. The cholinium based polymer systems with combined AMP¯ and CLX¯, show promise for antibiotic treatment, enabling the simultaneous co-delivery of drugs for optimal therapeutic outcomes. Declarations Conflicts of Interest: The authors declare no conflict of interest. Funding: The funding for these studies was provided by the Grant for Young Scientists BKM-123/RCH4/2024 (04/040/BKM24/0123) S.K. and Pro-Quality Rector's Grant 04/040/RGJ23/0244 D.N. Author Contribution S.K.: Investigation, Data curation, Formal analysis, Writing—original draft; D.N.: Conceptualization, Methodology, Writing—review and editing, Supervision. All authors have read and agreed to the published version of the manuscript. Data Availability All data generated or analysed during this study are included in this published article. References W. 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Ionic exchange of TMAMA/Cl with sodium salts of CLX and AMP to produce pharmaceutically functionalized choline monomers. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4580822","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":321709358,"identity":"56ed73a1-c272-417f-9826-43cb4c361aa4","order_by":0,"name":"Shadi Keihankhadiv","email":"","orcid":"","institution":"Silesian University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Shadi","middleName":"","lastName":"Keihankhadiv","suffix":""},{"id":321709359,"identity":"bb1f11e6-8203-4275-9682-c3ad7a980fb5","order_by":1,"name":"Dorota Neugebauer","email":"data:image/png;base64,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","orcid":"","institution":"Silesian University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Dorota","middleName":"","lastName":"Neugebauer","suffix":""}],"badges":[],"createdAt":"2024-06-14 09:02:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4580822/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4580822/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59611610,"identity":"83fed50d-4bde-4326-9119-a835a7da2b49","added_by":"auto","created_at":"2024-07-03 20:38:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":170651,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic route from the modification of choline IL by pharmaceutical anions CLX and AMP to the linear polymers as the single and dual systems, including drug release in PBS at 37\u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4580822/v1/e729e9146f34af2b90cbc4d5.png"},{"id":59610718,"identity":"2c8150a1-81b1-41ad-be77-e88b48951342","added_by":"auto","created_at":"2024-07-03 20:14:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":122733,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003csup\u003e1\u003c/sup\u003eH–NMR spectra of TMAMA/Cl before anion exchange (a) to the monomers TMAMA/AMP (b), and TMAMA/CLX (c).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4580822/v1/2e1b428b7b84d1682fb54f7f.png"},{"id":59610716,"identity":"e13a3571-0bc4-4ab8-98a6-83027765835b","added_by":"auto","created_at":"2024-07-03 20:14:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":73251,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH–NMR spectra of the reaction mixture at the end of the polymerization in the synthesis of single drug system IA (P(TMAMA/CLX–\u003cem\u003eco\u003c/em\u003e–MMA)) (M1 and M2 are related to the signals of TMAMA/CLX and MMA, respectively).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4580822/v1/554d56108b5acac4f3bb31cd.png"},{"id":59610721,"identity":"83bec732-a857-406d-b0ad-030571bac9ba","added_by":"auto","created_at":"2024-07-03 20:14:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":112333,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH-NMR spectra of the reaction mixture at the end of the polymerization in the synthesis of dual drug system IIA, (P(TMAMA/CLX–\u003cem\u003eco\u003c/em\u003e–MMA–\u003cem\u003eco\u003c/em\u003e–TMAMA/AMP)) (M1 and M2 are related to the signals of TMAMA and MMA, respectively).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4580822/v1/139a78b7c003fd5c470b432a.png"},{"id":59611459,"identity":"732ef805-f6b4-432e-9091-225641d89581","added_by":"auto","created_at":"2024-07-03 20:30:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":64356,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation between drug content (DC) and polymer length chain (DP\u003csub\u003en\u003c/sub\u003e) and amount of TMAMA units (DP\u003csub\u003eM1\u003c/sub\u003e) in single (a) and dual (b) systems.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4580822/v1/b4f608aab65695de4e9c0462.png"},{"id":59610726,"identity":"d62bb48b-feb0-442e-8a78-513d34cd4d4a","added_by":"auto","created_at":"2024-07-03 20:14:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":53646,"visible":true,"origin":"","legend":"\u003cp\u003eParticle size histograms for single (a) and dual (b) drug polymer systems by DLS analysis.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4580822/v1/5cd27fde53afad8a70f99b29.png"},{"id":59611460,"identity":"c4039fa6-aac0-48b2-bc01-79082463296d","added_by":"auto","created_at":"2024-07-03 20:30:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":130975,"visible":true,"origin":"","legend":"\u003cp\u003eDrug released data after 72 h for polymer conjugate systems by UV-Vis. ARD is amount of released drug (a), and CRD is concentration of released drug (b).\u003c/p\u003e","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4580822/v1/c345a88cae37eac4a9634869.png"},{"id":59610725,"identity":"d93f1232-3cb5-4fcd-a7de-afee4dc36891","added_by":"auto","created_at":"2024-07-03 20:14:43","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":74092,"visible":true,"origin":"","legend":"\u003cp\u003eKinetic release profiles of CLX\u003csup\u003e¯ \u003c/sup\u003efrom single drug systems (a) and CLX¯ and AMP\u003csup\u003e \u003c/sup\u003e¯ from dual drug systems (b, c) based on linear polymer conjugates.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-4580822/v1/dbafcdff41b85cf280900d45.png"},{"id":60470258,"identity":"e744f45a-5f5f-4d8a-88d6-9943ea855936","added_by":"auto","created_at":"2024-07-17 06:29:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1626668,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4580822/v1/89ed6e60-fc2b-4441-8e2e-3d42a867ba1b.pdf"},{"id":59611060,"identity":"9f0f4712-fe95-49fc-b86c-86493510d4be","added_by":"auto","created_at":"2024-07-03 20:22:43","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":193066,"visible":true,"origin":"","legend":"\u003cp\u003eGA\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4580822/v1/4abf682e736f93031af36118.png"},{"id":59611058,"identity":"702c455f-8787-4020-8099-592016d4d9cd","added_by":"auto","created_at":"2024-07-03 20:22:43","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":66476,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 1. Ionic exchange of TMAMA/Cl with sodium salts of CLX and AMP to produce pharmaceutically functionalized choline monomers.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4580822/v1/76d79393af3103c7a82a41b2.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Linear copolymers based on cholinium functionalized with antibiotic anions for single– and dual–drug delivery systems","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCholine, identified as 2-hydroxyethyl trimethylammonium chloride, is naturally synthesized by the human liver and is present in phospholipids like phosphatidylcholine or lecithin. This organic salt represents ionic liquids (ILs) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], which possess chemical stability, ability to enhance solubility, and modifiability by ion exchange for adjustment of physical and chemical properties [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Their biological attributes include enhancing skin penetration [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], acting as antibacterial properties [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], functioning as stabilizers [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], cytotoxic and local anesthetic properties, anti-fungal and anti-acne activities, and antibiotic actions [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Furthermore, the versatility of ILs extends to their ability to accommodate a wide range of pharmaceutical substances, including antiviral and antimicrobial agents, antioxidants, anticoagulants, nonsteroidal anti-inflammatory drugs, anticancer drugs, and others [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The widely employed cholinium cation offers biodegradability, water-solubility, and low cost for various applications [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Its properties have been investigated in combination with various bioactive compounds, including phenytoin [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], ampicillin (AMP) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], nalidixic acid, niflumic acid, \u003cem\u003ep\u003c/em\u003e-aminosalicylic acid, pyrazinoic acid, and picolinic acid [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These systems have demonstrated enhanced solubility for the active pharmaceutics, elevating their capability to permeate the cell membrane [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn recent decades, the synthesis of precisely designed polymers with the desired architecture, composition, chain homogeneity, site-specific functionality [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], physicochemical and biochemical attributes (e.g. mechanical strength, softness, self-healing, processability, tissue adhesiveness, bioactivity, and biodegradation) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] appeared to be a powerful material enabling the development of versatile nanostructures applicable in biology and medicine [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The tailored-made polymers synthesized by the controlled polymerization methods had influenced on significant progress in drug delivery systems (DDS) offering linear and branched polymer carriers with bio-therapeutics functions [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Both, drug conjugated or encapsulated by polymers have been intensively studied to address challenges related to the drug's hydrophilicity [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Furthermore, the delivery of more than one bioactive compound has been tested to enhance the main drug's activity [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe commercial choline ester derivative, [2-(methacryloyloxy)ethyl]trimethyl-ammonium chloride, referred to methacroylcholine (TMAMA/Cl), functions as a choline-based ionic liquid, which serves as the monomer for obtaining the polymerized ionic liquid (PIL) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. This PIL has been reported as delivering pharmaceutical anions through the anion exchange in the polymer matrix[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] or encapsulating various bioactive compounds [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] to create a pharmaceutically active polymeric systems. The pharmaceutically active choline-based PILs have been also designed by direct polymerization of pharmaceutically functionalized choline monomers with salicylate [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], \u003cem\u003ep\u003c/em\u003e-aminosalicylate [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], fusidate [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], and cloxacillin (CLX)[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] counterions.\u003c/p\u003e \u003cp\u003eIn this research, we investigated the well-defined linear copolymers based on the biofunctionalized choline ionic liquids with ionically conjugated AMP and CLX as the pharmaceutical anions. Depending on the strategy employed, the linear copolymers were designed either as single DDS carrying CLX\u0026macr; or dual DDS with CLX\u0026macr; and AMP\u0026macr; (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), where the drug anion is linked via an ionic bond to a polymer matrix. Both drugs are antibiotics deriving from semi-synthetic penicillin, which demonstrates antimicrobial efficacy from the existence of a beta-lactam ring [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Because of effectiveness against both gram-positive and some gram-negative microorganisms [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] they are employed for the treatment of bacterial infections affecting the ear, nose, throat, bones, lungs, as well as post-operative wound infections [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The commercially available combination of AMP and CLX is marketed under various brand names, such as Ampiclox, Apen, Cloxam, and Megamox, in the form of capsules or oral suspensions. Therefore, both pharmaceuticals are well-suited for utilization in either individual treatment or combined therapies. The obtained polymer systems were characterized to show their effectiveness in drug delivery through the evaluation of drug content and (co)release during an \u003cem\u003ein vitro\u003c/em\u003e study. The advantage of this novel DDSs is featured by the selected antibiotics carried by the ionic polymers, where pharmaceutical anions are released by physiological solution by exchange with phosphate anions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003eMethyl methacrylate (MMA, Alfa Aesar, Warsaw, Poland), tetrahydrofuran (THF, Sigma Aldrich, Poznan, Poland) and methanol (MeOH, Chempur, Piekary Śląskie, Poland) were dried using molecular sieves (type 4A, bulk density 640\u0026ndash;670 kg/m\u003csup\u003e3\u003c/sup\u003e, Chempur, Piekary Śląskie, Poland) under argon. [2-(Methacryloyloxy)ethyl]trimethylammonium chloride (TMAMA/Cl, 80% aq. solution, Sigma-Aldrich, Poznan, Poland) was concentrated under vacuum until it achieved solid product. Copper (I) bromide (CuBr, Fluka, Steinheim, Germany) was purified by stirring in glacial acetic acid, followed by filtration, and washing with ethanol and diethyl ether, and subsequent vacuum drying. Deionized water was obtained using the Hydrolab HLP Uv5 equipment (Straszyn, Poland). Ethyl 2-bromoisobutyrate (EBiB), phosphate-buffered saline (PBS, pH 7.4), \u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e\u0026prime;,\u003cem\u003eN\u003c/em\u003e\u0026Prime;,\u003cem\u003eN\u003c/em\u003e\u0026Prime;-pentamethyldiethylenetriamine (PMDETA) and ampicillin sodium salt (NaAMP) from Sigma Aldrich (Poznan, Poland), cloxacillin sodium monohydrate (NaCLX) and deuterated dimethyl sulfoxide (DMSO\u0026ndash;d6) from Alfa Aesar, as well as \u003cem\u003eN,N\u003c/em\u003e-dimethylforamide (DMF, POCH, Gliwice, Poland), phosphate buffer solution (PBS, Sigma Aldrich, Poznan, Poland) and diethyl ether (Chempur, Piekary Śląskie, Poland), were used without prior purification.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Preparation of pharmaceutically functionalized choline ionic liquids by ion exchange\u003c/h2\u003e \u003cp\u003eThe vacuum dried TMAMA/Cl (2.14 mmol, 0.445 g) was dissolved in 2.2 mL of MeOH (forming solution 1). Next, NaCLX (2.14 mmol, 1.02 g) was dissolved in 5.1 mL of MeOH (TMAMA/Cl: MeOH\u0026thinsp;=\u0026thinsp;1:5 w/v) and added dropwise to solution 1 while the mixture stirred continuously during drug addition. Then, the mixture was stirred for 3 hours constantly in a dark place at room temperature during the ion exchange reaction. After NaCl salt precipitation, the solution was filtered and washed twice with 1 mL MeOH to remove any salt. To accelerate MeOH evaporation, the filtrated solution containing TMAMA/CLX was left on a ventilator at room temperature for 30 minutes. It was further dried under vacuum until it solidified into a powder product. Yield: 1.26 g. \u003csup\u003e1\u003c/sup\u003eH NMR (DMSO\u0026ndash;d\u003csub\u003e6\u003c/sub\u003e,300 MHz, δ, ppm): 7.4\u0026ndash;7.7 (4H, aromatic ring of CLX), 5.75 and 6.15 (2H, =CH\u003csub\u003e2\u003c/sub\u003e in vinyl group), 5.25\u0026ndash;5.45 (2H, -CH-N- and -CH-S- in β-lactam ring of CLX), 4.54 (2H, -CH\u003csub\u003e2\u003c/sub\u003e-O-), 3.82 (1H, -CH-N- in thiazolidine ring of CLX), 3.73 (2H, -CH\u003csub\u003e2\u003c/sub\u003e-N\u003csup\u003e+\u003c/sup\u003e-), 3.14 (9H, -N\u003csup\u003e+\u003c/sup\u003e(CH\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e), 2.65 (3H, -CH\u003csub\u003e3\u003c/sub\u003e at isoxazole ring in CLX), 1.92 (3H, -CH\u003csub\u003e3\u003c/sub\u003e), 1.46 (6H, -C(CH\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e in thiazolidine ring of CLX).\u003c/p\u003e \u003cp\u003eThe synthesis of analogical TMAMA/AMP followed the previously described method, utilizing equimolar ratios of TMAMA/Cl (3.9 mmol, 0.8 g) and AMPNa (3.9 mmol, 1.43 g) in MeOH (4 mL and 7.2 mL, respectively). However, the ion exchange process was completed within 20 hours. Yield: 2.04 g. \u003csup\u003e1\u003c/sup\u003eH\u0026ndash;NMR (DMSO-d\u003csub\u003e6\u003c/sub\u003e,300 MHz, δ, ppm): 7.1\u0026ndash;7.5 (5H, -CH in aromatic ring in AMP), 5.75 and 6.15 (2H, -CH\u003csub\u003e2\u003c/sub\u003e in vinyl group), 5.25\u0026ndash;5.5 (2H, -CH-N- and -CH-S- in β-lactam ring of AMP), 4.54 (2H, -CH\u003csub\u003e2\u003c/sub\u003e-O-), 4.46 (1H, -CH-NH\u003csub\u003e2\u003c/sub\u003e in AMP), 3.8-4.0 (1H, -CH-N- in thiazolidine ring of AMP), 3.75 (2H, -CH\u003csub\u003e2\u003c/sub\u003e-N\u003csup\u003e+\u003c/sup\u003e-), 3.16 and 3.12 (9H, -N\u003csup\u003e+\u003c/sup\u003e(CH\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e), 1.92 (3H, -CH\u003csub\u003e3\u003c/sub\u003e), 1.57 and 1.46 (6H, -C(CH\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e in thiazolidine ring of AMP).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Synthesis of copolymer conjugates\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e2.3.1. Ionic linear copolymers containing CLX anion, P(TMAMA/CLX\u003c/b\u003e\u0026ndash;\u003cb\u003eco\u003c/b\u003e\u0026ndash;\u003cb\u003eMMA) (Example for IA)\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTMAMA/CLX (0.656 mmol, 0.41 g) was dissolved in 0.41 mL of MeOH in a Schlenk flask. Then, MMA (1.968 mmol, 0.21 mL), THF (0.14 mL), and PMDETA (0.007 mmol, 0.001 mL) were added to the flask. The mixture underwent homogenization and degassing by two freeze\u0026ndash;pump\u0026ndash;thaw cycles. Then, EBiB (0.007 mmol, 0.001 mL) was introduced as an initiator, and the initial sample was taken. Another freeze\u0026ndash;pump\u0026ndash;thaw cycle was performed. Following this, CuBr catalyst (0.007 mmol, 0.001 g) was quickly added to the mixture, and the reaction flask was immersed in an oil bath at 40\u0026deg;C. After 2 hours, when a noticeable increase in the mixture's viscosity was observed, the reaction was stopped by exposing it to air. Subsequently, the resulting product was dissolved in MeOH (1 mL) and precipitated twice in a chloroform\u0026ndash;diethyl ether mixture (1:1) to eliminate the catalyst from the polymer. Solvents were discarded using a syringe, and the remaining solvents were evaporated under ventilator at room temperature for 15 minutes. Finally, the polymer was dried under the vacuum. Yield: 0.24 g. \u003csup\u003e1\u003c/sup\u003eH\u0026ndash;NMR (DMSO\u0026ndash;d\u003csub\u003e6\u003c/sub\u003e, 300 MHz, δ, ppm): 7.4\u0026ndash;7.7 (4H, aromatic ring of CLX), 5.3-5,5 (2H, -CH-N- and -CH-S- in β-lactam ring of CLX), 4.1\u0026ndash;4.3 (2H, -CH\u003csub\u003e2\u0026minus;\u003c/sub\u003eO-), 3.82 (1H, -CH-N- in thiazolidine ring of CLX), 3.60\u0026ndash;3.75 (2H, -CH\u003csub\u003e2\u003c/sub\u003e-N\u003csup\u003e+\u003c/sup\u003e-), 3.5\u0026ndash;3.6 (3H, -O-CH\u003csub\u003e3\u003c/sub\u003e), 3.02\u0026ndash;3.30 (9H, -N\u003csup\u003e+\u003c/sup\u003e(CH\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e), 2.65 (3H, -CH\u003csub\u003e3\u003c/sub\u003e at isoxazole ring in CLX), 1.75 (2H, -CH\u003csub\u003e2\u003c/sub\u003e-C-), 1.36\u0026ndash;1.46 (6H, -C(CH\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e in thiazolidine ring of CLX), 0.54\u0026ndash;1.2 (3H, -CH\u003csub\u003e3\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e2.3.2. Dual bioactive ionic linear copolymers containing CLX\u0026macr;/AMP\u0026macr;, P(TMAMA/CLX\u003c/b\u003e\u0026ndash;\u003cb\u003eco\u003c/b\u003e\u0026ndash;\u003cb\u003eMMA\u003c/b\u003e\u0026ndash;\u003cem\u003eco\u003c/em\u003e\u0026ndash;\u003cb\u003eTMAMA/AMP) (Example for IIA)\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTMAMA/CLX (0.557 mmol, 0.35 g) and TMAMA/AMP (0.557 mmol, 0.29 g) were dissolved in 0.64 mL of MeOH in a Schlenk flask. Then, MMA (3.342 mmol, 0.36 mL), THF (0.21 mL), and PMDETA (0.011 mmol, 0.002 mL) were added to the flask. Further steps were provided like for synthesis and purification of P(TMAMA/CLX\u0026ndash;\u003cem\u003eco\u003c/em\u003e\u0026ndash;MMA) described in section \u003cspan refid=\"Sec5\" class=\"InternalRef\"\u003e2.3.1\u003c/span\u003e using proper amounts of EBiB (0.011 mmol, 0.002 mL) and CuBr (0.011 mmol, 0.002 g). Yield: 0.73 g. \u003csup\u003e1\u003c/sup\u003eH\u0026ndash;NMR (DMSO\u0026ndash;d\u003csub\u003e6\u003c/sub\u003e,300 MHz, δ, ppm): 7.1\u0026ndash;7.7 (9H, -CH in aromatic rings of AMP and CLX), 5.25\u0026ndash;5.4 (2H, -CH-NH- and -CH-S- in β-lactam ring of CLX and AMP), 4.43 (1H, -CH-NH\u003csub\u003e2\u003c/sub\u003e in AMP), 4.1\u0026ndash;4.3 (2H, -CH\u003csub\u003e2\u0026minus;\u003c/sub\u003eO-), 3.75\u0026ndash;3.87 (2H, -CH-N- in thiazolidine ring of CLX and AMP), 3.65\u0026ndash;3.75 (2H, -CH\u003csub\u003e2\u003c/sub\u003e-N\u003csup\u003e+\u003c/sup\u003e-), 3.4\u0026ndash;3.65 (3H, -O-CH\u003csub\u003e3\u003c/sub\u003e), 3.05\u0026ndash;3.25 (9H, -N\u003csup\u003e+\u003c/sup\u003e(CH\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e), 2.65 (3H, -CH\u003csub\u003e3\u003c/sub\u003e at isoxazole ring in CLX), 1.75 (2H, -CH\u003csub\u003e2\u003c/sub\u003e-C), 1.4\u0026ndash;1.7 (12H, -C(CH\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e in thiazolidine ring of CLX and AMP), 0.6\u0026ndash;1.2 (3H, -CH\u003csub\u003e3\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Release studies of copolymer conjugates containing CLX\u0026macr; and AMP\u0026macr;\u003c/h2\u003e \u003cp\u003eCopolymer conjugates containing drug anions (1 mg) were dissolved in 1 mL of PBS (pH 7.4) to achieve a 1 mg/mL solution. The solution was then transferred into a dialysis cellulose membrane bag with a molecular weight cutoff (MWCO) of 3.5 kDa. Then, this membrane bag was placed inside a glass vial filled with 44 mL of PBS and stirred at 37\u0026deg;C during a 74\u0026ndash;hour dialysis process. The release progress was monitored by assessing changes in drug concentration in the external PBS solution surrounding the dialysis bag. At different time intervals, 0.5 mL samples of the solution containing the released drug were collected, and 0.5 mL of MeOH was added to the cuvette. These samples were subsequently analyzed using UV-Vis spectrophotometer to quantify the released drug amount by measuring the absorbance at a wavelength of λ\u0026thinsp;=\u0026thinsp;206 nm for AMP\u0026macr; and λ\u0026thinsp;=\u0026thinsp;203 nm for CLX\u0026macr;. Each reported result represents the average of three measurements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Characterization methods\u003c/h2\u003e \u003cp\u003eProton nuclear magnetic resonance (\u003csup\u003e1\u003c/sup\u003eH\u0026ndash;NMR) spectra were acquired using a UNITY/NOVA spectrometer (Varian, Mulgrave, Victoria, Australia) operating at frequency of 300 MHz. The measurements were conducted on samples dissolved in DMSO\u0026ndash;d\u003csub\u003e6\u003c/sub\u003e with tetramethylsilane used as the internal standard. For size exclusion chromatography (SEC), an Ultimate 3000 chromatograph (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a differential refractometer detector RefractoMax 521 was employed. The polymer samples prepared in DMF containing 10 mM LiBr at 40\u0026deg;C, were passed through a precolumn TSKgel Guardian SuperMP(HZ)-H (4.6 mm \u0026times; 2 cm, with a particle size of 6 \u0026micro;m), followed by two columns of TSKgel SuperMilipore HZ-H (4.6 mm \u0026times; 15 cm, with a particle size of 6 \u0026micro;m). The flow rate was maintained at 0.45 mL/min. The average molecular weight (M\u003csub\u003en\u003c/sub\u003e) and dispersity index (\u0026ETH;) were determined on the base of poly(ethylene oxide)/poly(ethylene glycol) (PEO) standards with molecular weights ranging from 982 to 969,000 g/mol. The drug content (DC) and the amount of released drug (ARD) were analyzed by ultraviolet-visible light spectroscopy (UV-Vis) employing a spectrometer Evolution 300 (Thermo Fisher Scientific, Waltham, MA, USA). Sample of the polymer dissolved in PBS and MeOH (1:1) at a concentration of 0.05 mg/mL were placed in a quartz cuvette and measured at a maximum wavelength of λ\u0026thinsp;=\u0026thinsp;206 nm for AMP\u0026macr; and λ\u0026thinsp;=\u0026thinsp;203 nm for CLX\u0026macr;. A calibration curve was established for drug concentrations ranging from 0.1 mg/mL to 0.006 mg/mL in the mix of PBS and MeOH (1:1). Dynamic light scattering (DLS) was performed using Nanotrac Flex with laser particle size analyzer Mictrac MRB's (Microtrac Retsch GmbH, Haan, Germany; Dimensions LS software 1.1.0.), equipped with an external \u0026ldquo;dip-in\u0026rdquo; probe with 180\u003csup\u003e0\u003c/sup\u003e backscattering. Polymer samples were dissolved in deionized water at a concentration of 1.0 mg/mL, and measurements were repeated for each sample at least three times to analyze the hydrodynamic diameters (Dh) of polymer nanoparticles and their polydispersity index (PDI).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Result and Discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Monomeric ionic liquid functionalized by pharmaceutical anions\u003c/h2\u003e \u003cp\u003eThe proposed strategy to pharmaceutically functionalized copolymers included the modification of the water-soluble ionic liquid, that is [2-(methacryloyloxy)ethyl]-trimethylammonium chloride (TMAMA/Cl) with polymerizable methacrylate group and Cl\u0026macr;, where the latter one was exchanged by CLX\u0026macr; and AMP\u0026macr; becoming from their sodium salts (NaCLX and NaAMP). This exchange reaction resulted in a monomers carrying a pharmaceutical anion, namely [2-(methacryloyloxy)ethyl]trimethylammonium cloxacillin (TMAMA/CLX) and ampicillin (TMAMA/AMP) as depicted in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The monomer structures were confirmed by the \u003csup\u003e1\u003c/sup\u003eH-NMR spectra to evaluate the efficiency of ion exchange by assigning characteristic proton signals to TMAMA, especially 9 protons in the trimethylammonium cation TMAMA units (D), and the pharmaceutical counterions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In the case of TMAMA/AMP the signal D at 3.16 ppm was still existing after exchange, but a new one appeared at 3.12 ppm, which demonstrated a 47% of exchange efficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), whereas for TMAMA/CLX it was slightly shifted to 3.14 ppm indicating 100% of the anion exchange (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Linear polymers containing CLX\u0026macr; as single drug delivery systems\u003c/h2\u003e \u003cp\u003eThe above-described ionic monomer TMAMA/CLX, with a choline species and pharmaceutically active counterion was copolymerized with the non-ionic monomer MMA in molar ratios of 25/75, 50/50, and 75/25 to synthesize the well-defined linear copolymers, denoted as P(TMAMA/CLX\u0026ndash;\u003cem\u003eco\u003c/em\u003e\u0026ndash;MMA)s (IA\u0026ndash;IC) as the single drug systems carrying different ionic content of TMAMA/CLX. For this purpose, the ATRP reactions were initiated by EBiB as a monofunctional initiator, catalyzed by CuBr/PMDETA complex, dissolved in MeOH/THF solvent mixture, and conducted at 40\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The conversion of the monomer to polymer and polymer structure were confirmed by \u003csup\u003e1\u003c/sup\u003eH\u0026ndash;NMR spectroscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The calculation of total conversions of both monomers (X) was relied on a broad signal deriving from protons of the methyl group in the formed polymer backbone (B, 0.54\u0026ndash;1.2 ppm) in relation to the signals from a proton in the vinyl groups of unreacted TMAMA and MMA monomers (identified as M1 and M2, in the range of 5.25 to 6.25 ppm). Moreover, the proton signal in the vinyl group of the unreacted monomer M1 at 6.09 ppm and the proton signal associated with the trimethylammonium group present in both the monomer and resulting polymer (referred to F at 3.02\u0026ndash;3.30 ppm) were utilized to determine the TMAMA conversion (X\u003csub\u003eM1\u003c/sub\u003e). Further, the conversion values were utilized to calculate other parameters, such as polymerization degree (DP), the ionic fraction content in the copolymer (F\u003csub\u003eM1\u003c/sub\u003e), and the average molecular weight of the copolymer (M\u003csub\u003en\u003c/sub\u003e), which are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of linear copolymer P(TMAMA/CLX\u0026ndash;\u003cem\u003eco\u003c/em\u003e\u0026ndash;MMA)\u0026rsquo;s synthesized by ATRP.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ef\u003csub\u003eM1\u003c/sub\u003e/f\u003csub\u003eM2\u003c/sub\u003e (mol%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTime (h)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eX\u003csub\u003eM1\u003c/sub\u003e\u003csup\u003ea\u003c/sup\u003e (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eX\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDP\u003csub\u003eM1\u003c/sub\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eDP\u003csub\u003en\u003c/sub\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eF\u003csub\u003eM1\u003c/sub\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eM\u003csub\u003en\u003c/sub\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(g/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eM\u003csub\u003en\u003c/sub\u003e\u003csup\u003eb\u003c/sup\u003e (g/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026ETH;\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25/75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e59900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e10700\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIB\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50/50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e179\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e241\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e115100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75/25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e277\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e299\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e170200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWhere: M1\u0026thinsp;=\u0026thinsp;TMAMA/CLX, M2\u0026thinsp;=\u0026thinsp;MMA; conditions: [TMAMA/CLX\u0026thinsp;+\u0026thinsp;MMA]\u003csub\u003e0\u003c/sub\u003e: [EBiB]\u003csub\u003e0\u003c/sub\u003e: [CuBr]\u003csub\u003e0\u003c/sub\u003e: [PMDETA]\u003csub\u003e0\u003c/sub\u003e= 400:1:1:1; MeOH:TMAMA/CLX\u0026thinsp;=\u0026thinsp;1:1 υ/ωt, MeOH:THF\u0026thinsp;=\u0026thinsp;3:1 υ/υ, 40\u003csup\u003eo\u003c/sup\u003eC; f\u003csub\u003eM1\u003c/sub\u003e, f\u003csub\u003eM2\u003c/sub\u003e \u0026ndash; initial content of monomer in the reaction mixture; X\u003csub\u003eM1\u003c/sub\u003e\u003csup\u003ea\u003c/sup\u003e \u0026ndash;TMAMA conversion; X - total monomer conversion; DP\u003csub\u003eM1\u003c/sub\u003e - polymerization degree of ionic monomers; DP\u003csub\u003en\u003c/sub\u003e - total polymerization degree; F\u003csub\u003eM1\u003c/sub\u003e - content of ionic fraction in the polymer; M\u003csub\u003en\u003c/sub\u003e - average molecular weight; Đ \u0026ndash; dispersity index. \u003csup\u003ea\u003c/sup\u003edetermined by \u003csup\u003e1\u003c/sup\u003eH-NMR (DMSO-d6), \u003csup\u003eb\u003c/sup\u003edetermined by SEC (DMF, PEO calibration).\u003c/p\u003e \u003cp\u003eLinear copolymers were obtained within a short time of 2 h by total monomer conversions ranging from 40\u0026ndash;75%, while the conversion of ionic monomer remained similar (87\u0026ndash;92%). The observed tendency of the monomer conversion increases with the initial content of TMAMA monomer shows that the latter factor can be used to regulate the length of polymer chain (DP\u003csub\u003en\u003c/sub\u003e=160\u0026ndash;299) and the amount of ionic units (DP\u003csub\u003eM1\u003c/sub\u003e=87\u0026ndash;277). The compositions of the resulting copolymers IA\u0026ndash;IC revealed a higher content of the ionic fraction than it was anticipated by the initial content of the ionic monomer (f\u003csub\u003eM1\u003c/sub\u003e/F\u003csub\u003eM1\u003c/sub\u003e: 25/54 (IA); 50/74 (IB); 75/93 (IC)). This phenomenon indicates higher reactivity of the ionic monomer (TMAMA/CLX), which is favored over MMA under polar conditions, probably leading to the formation of copolymers with gradient structures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Linear polymers containing CLX\u0026macr;/AMP\u0026macr; as dual drug delivery systems\u003c/h2\u003e \u003cp\u003eThe dual drug anions carried by polymer conjugates resulted by the terpolymerization of two pharmaceutically functionalized choline monomers, i.e. both TMAMA/CLX and TMAMA/AMP with MMA in the ratio equal to 12.5:12.5:75, 25:25:50, 37.5:37.5:25 via ATRP under conditions similar to those above described for the synthesis of single drug copolymer conjugates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The P(TMAMA/CLX\u0026ndash;\u003cem\u003eco\u003c/em\u003e\u0026ndash;MMA\u0026ndash;\u003cem\u003eco\u003c/em\u003e\u0026ndash;TMAMA/AMP)s (IIA\u0026ndash;IIC, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) were synthesized to be capable of transporting two drugs concurrently within the system. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the assessment of monomers converted into the polymers through \u003csup\u003e1\u003c/sup\u003eH\u0026ndash;NMR analysis, employing analogous signals as previously described for series IA\u0026ndash;IC.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of linear terpolymers P(TMAMA/CLX\u0026ndash;\u003cem\u003eco\u003c/em\u003e\u0026ndash;MMA\u0026ndash;\u003cem\u003eco\u003c/em\u003e\u0026ndash;TMAMA/AMP)\u0026rsquo;s synthesized by ATRP.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ef\u003csub\u003eM1\u003c/sub\u003e/f\u003csub\u003eM2\u003c/sub\u003e (mol%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTime (h)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eX\u003csub\u003eM1\u003c/sub\u003e\u003csup\u003ea\u003c/sup\u003e (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eX\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDP\u003csub\u003eM1\u003c/sub\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eDP\u003csub\u003en\u003c/sub\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eF\u003csub\u003eM1\u003c/sub\u003e\u003csup\u003ea\u003c/sup\u003e (mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eM\u003csub\u003en\u003c/sub\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(g/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eM\u003csub\u003en\u003c/sub\u003e\u003csup\u003eb\u003c/sup\u003e (g/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026ETH;\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIIA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25/75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e239\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e55500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e16200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIIB\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50/50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e181\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e85400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIIC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75/25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e275\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e124800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWhere: M1\u0026thinsp;=\u0026thinsp;TMAMA/CLX\u0026thinsp;+\u0026thinsp;TMAMA/AMP, M2\u0026thinsp;=\u0026thinsp;MMA; conditions: [(TMAMA/CLX\u0026thinsp;+\u0026thinsp;TMAMA/AMP)\u0026thinsp;+\u0026thinsp;MMA]\u003csub\u003e0\u003c/sub\u003e: [EBiB]\u003csub\u003e0\u003c/sub\u003e: [CuBr]\u003csub\u003e0\u003c/sub\u003e: [PMDETA]\u003csub\u003e0\u003c/sub\u003e= 400:1:1:1; MeOH:TMAMA\u0026thinsp;=\u0026thinsp;1:1 υ/ωt, MeOH:THF\u0026thinsp;=\u0026thinsp;3:1 υ/υ, 40\u003csup\u003eo\u003c/sup\u003eC; f\u003csub\u003eM1\u003c/sub\u003e, f\u003csub\u003eM2\u003c/sub\u003e \u0026ndash; initial content of comonomers in the reaction mixture; X\u003csub\u003eM1\u003c/sub\u003e\u003csup\u003ea\u003c/sup\u003e \u0026ndash; conversion of both TMAMA monomers; X - total conversion; DP\u003csub\u003eM1\u003c/sub\u003e - polymerization degree of ionic monomers; DP\u003csub\u003en\u003c/sub\u003e - total polymerization degree; F\u003csub\u003eM1\u003c/sub\u003e - content of ionic fraction in the polymer; M\u003csub\u003en\u003c/sub\u003e - average molecular weight of copolymer; Đ \u0026ndash; dispersity index. \u003csup\u003ea\u003c/sup\u003edetermined by \u003csup\u003e1\u003c/sup\u003eH-NMR (DMSO-d6), \u003csup\u003eb\u003c/sup\u003edetermined by SEC (DMF, PEO calibration).\u003c/p\u003e \u003cp\u003eThe presence of the second drug based TMAMA in the polymerization with the lowest initial content of ionic monomers affected the formation of significantly higher chain lengths and similar amounts of incorporated ionic units when comparing IIA with IA (DP\u003csub\u003en\u003c/sub\u003e/DP\u003csub\u003eM1\u003c/sub\u003e = 239/91 vs 160/87). This observation suggests possible differences in the relative reactivities of ionic comonomers, where TMAMA with AMP counterion appearing to be more reactive than that with CLX anion, which presents higher steric hindrance resulting from its Cl-substituted aromatic ring and extra 5-membered ring. Additionally, due to its structure, CLX seems to be less hydrophilic than AMP, making TMAMA/AMP more favorable under the polymerization conditions used. In relation to MMA, both TMAMAs polymerized faster, as evidenced by the correlation F\u003csub\u003eM1\u003c/sub\u003e \u0026gt; f\u003csub\u003eM1\u003c/sub\u003e. In the system of three monomer components, MMA was converted at a higher rate than in two monomer system, leading to lower ionic content in polymer IIA compared to IA (38% vs 54%). However, a higher initial concentration of TMAMAs (f\u003csub\u003eM1\u003c/sub\u003e = 50 and 75%) provided similar total monomer conversions, as well as DP\u003csub\u003en\u003c/sub\u003e and DP\u003csub\u003eM1\u003c/sub\u003e values, to analogous systems in series I, but it required longer time of polymerization as convenient for incorporation of slower TMAMA/CLX. Generally, knowing these relations the compositions of the resulting polymers IIA\u0026ndash;IIC can be assumed by the initial proportions of TMAMA/MMA (f\u003csub\u003eM1\u003c/sub\u003e/F\u003csub\u003eM1\u003c/sub\u003e: 25/38 (IIA); 50/78 (IIB); 75/92 (IIC)).\u003c/p\u003e \u003cp\u003eThe SEC data were obtained for copolymers with ionic fraction below 60%, which were well-soluble in DMF. verifying the well-controlled polymerization. The molecular weight distribution for the dual drug polymer IIA was larger than for the single drug IA (Đ=1.58 vs. 1.24), which suggests that the occurrence of side reactions was promoted in the system containing two types of TMAMA counterions with different natures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe drug content (DC), which defines the percentage of pharmaceutical anions included in the polymer chain, was evaluated by UV-Vis spectroscopy. The DC of CLX\u0026macr; was ranged in 67\u0026ndash;80% in the single (IA\u0026ndash;IC) and 51\u0026ndash;64% in the dual drug systems (IIA\u0026ndash;IIC), where the latter ones also co-delivered 78\u0026ndash;87% of AMP\u0026macr; (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The simultaneous increase of DCs driven by the total polymerization degree, including polymerization degree of TMAMA was observed in the single drug polymers in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea (DC/DP\u003csub\u003en\u003c/sub\u003e/DP\u003csub\u003eM1\u003c/sub\u003e: 67/160/87 (IA), 73/241/179 (IB), 80/299/277 (IC)). Similarly, the trend of increasing DC correlated with the amount of TMAMA units was also indicated for the dual systems with additional relation between DC of both drugs, that is DC\u003csub\u003eCLX\u003c/sub\u003e \u0026lt; DC\u003csub\u003eAMP\u003c/sub\u003e (IIA: 51% CLX\u0026macr; vs 78% AMP\u0026macr;, IIB: 62% CLX\u0026macr; vs 83% AMP\u0026macr;, IIC: 64% CLX\u0026macr; vs 87% AMP\u0026macr;) as demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb. In summary, the highest values for CLX\u0026macr; were achieved for IC and IIC with the longest polymer chain lengths and the highest ionic contents (DP\u003csub\u003en\u003c/sub\u003e ~ 300 and F\u003csub\u003eM1\u003c/sub\u003e \u0026gt; 92%), which makes them the most advantageous systems.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Hydrodynamic diameters of polymer particles\u003c/h2\u003e \u003cp\u003eDynamic light scattering (DLS) analysis was conducted to assess the particle size of both types of drug delivery polymer conjugates in an aqueous solution (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The histograms in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e depict the distribution of the formed particles exhibiting one fraction. The results for the single drug polymer systems revealed distinct hydrodynamic diameters (Dh) for IA and IC exhibited Dh\u0026thinsp;\u0026le;\u0026thinsp;274 nm, whereas IB displayed a larger value of 380 nm. In the dual drug systems (IIA\u0026ndash;IIC), despite the differences in polymer structure, not big significant influence was observed on particle sizes, which ranged from 288 nm to 348 nm. Furthermore, the polydispersity index (PDI), which defines the level of heterogeneity of particles as the distribution of their sizes, indicated uniformity of particle sizes (PDI\u0026thinsp;\u0026le;\u0026thinsp;0.01). Remarkably narrow size distributions were exhibited by IB and IIC (PDI: 0.006 and 0.008, respectively).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDLS characteristics of linear polymer particles carrying drug.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType DDS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePolymer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDh (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePDI\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eSingle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e274\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e380\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e277\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eDual\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIIA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e288\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIIB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e324\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIIC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e348\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.008\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ewhere: Dh is hydrodynamic diameter and PDI is polydispersity index.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Drug Release\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e drug release studies were conducted to monitor the exchange of CLX\u003csup\u003e\u0026macr;\u003c/sup\u003e or CLX\u003csup\u003e\u0026macr;\u003c/sup\u003e/AMP\u003csup\u003e\u0026macr;\u003c/sup\u003e anions in the polymer matrix by phosphate anions in the PBS via the dialysis method under physiological conditions in pH 7.4 at 37\u0026deg;C for 72 hours. Drug release from the polymer samples was detected at specified time intervals, and the percentage amount of released drug (ARD, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea) correlated with the drug concentration in the released medium (CRD, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb) in relation to the DC were determined using UV\u0026ndash;Vis spectroscopy. The kinetic profiles for the single drug systems illustrated a release of 34\u0026ndash;41% of CLX\u0026macr; within 0.5\u0026ndash;1 hour, which increased to 58\u0026ndash;72% within four hours and continued up to 74 hours, representing relative stability with additional 4\u0026ndash;6% of drug release (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). However, in the case of sample IC the release profile differed from the others, exhibiting a more linear relationship that was initially similar to the system IA, but ultimately reached the highest ARD value. The increase in the amount of drug released from the single systems corresponded well with elevated total polymerization degree and drug content (DP\u003csub\u003en\u003c/sub\u003e/DC/ARD: 160/67/58 (IA), 241/73/66 (IB), 299/80/76 (IC)). Combining the effective content of drug and its release, polymer IC appeared to be the most promising for an extended therapeutic effect.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the context of the dual co-delivery systems, a significant initial burst release was observed in the first hour for both drugs, yielding 64\u0026ndash;80% for CLX\u0026macr; (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb) and 90\u0026ndash;98% for AMP\u0026macr; (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec). Following the release rate was notably slowed down yielding an additional 12\u0026ndash;16% of CLX\u0026macr; released within the next 2 hours, and reaching ARD values of 88\u0026ndash;100% after 4 hours. For incomplete release, the process was extended up to 74 hours, but slight progress was detected, maintaining a release plateau. In summary, complete release of both drugs was achieved by system IIA within 4 hours, whereas the release from IIB and IIC systems was slightly slower, reaching\u0026thinsp;\u0026gt;\u0026thinsp;90% of CLX\u0026macr; and \u0026gt;\u0026thinsp;97% of AMP\u0026macr;. The results for series II suggested that the interactions between CLX\u0026macr; and AMP\u0026macr; improved the co-release of CLX, although its DC was slightly lower than that in the series I, but the polymers, especially IIA, were able to completely release both drugs in a relatively short time.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThe well-defined linear copolymers, including series I with CLX\u0026macr; (single drug DDS) and series II containing both CLX\u0026macr; and AMP\u0026macr; (dual drug DDS), were synthesized to demonstrate their potential as nanocarriers (274\u0026ndash;380 nm (CLX\u0026macr;) and 288\u0026ndash;348 nm (CLX\u0026macr;/AMP\u0026macr;)). In both series, the TMAMA monomers functionalized by pharmaceutical anions with antibiotic activities were incorporated into the polymer chains in different contents (38\u0026ndash;93 mol%). The various lengths of polymer chains (DP\u003csub\u003en\u003c/sub\u003e=160\u0026ndash;300) were adjusted by the monomer conversion (40\u0026ndash;75%), while the initial proportion of comonomers allowed for regulation of ionic contents (38\u0026ndash;93 mol%) in the polymers.\u003c/p\u003e \u003cp\u003eThe drug content within the polymer matrix, ranging in 67\u0026ndash;80% in the CLX systems, and 51\u0026ndash;64% of CLX\u0026macr; and 78\u0026ndash;87% of AMP\u0026macr; in the dual drug systems, corresponded to efficient \u003cem\u003ein vitro\u003c/em\u003e release of 91\u0026ndash;100% of CLX\u0026macr; (12.9\u0026ndash;15.1 \u0026micro;g/mL) and 97\u0026ndash;100% of AMP\u0026macr; (21.1\u0026ndash;23.3 \u0026micro;g/mL) from the dual drug systems, and 67\u0026ndash;80% of CLX\u0026macr; in the single drug systems in PBS within 72h. The drug content and release were significantly affected by factors, such as polymer chain length, ionic fraction content in the copolymer, and pharmaceutical anion nature. Generally, linear copolymers demonstrated effectiveness in designing both single\u0026ndash; and dual\u0026ndash;drug systems with customized release profiles, facilitating drug delivery. The cholinium based polymer systems with combined AMP\u0026macr; and CLX\u0026macr;, show promise for antibiotic treatment, enabling the simultaneous co-delivery of drugs for optimal therapeutic outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of Interest:\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThe funding for these studies was provided by the Grant for Young Scientists BKM-123/RCH4/2024 (04/040/BKM24/0123) S.K. and Pro-Quality Rector's Grant 04/040/RGJ23/0244 D.N.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.K.: Investigation, Data curation, Formal analysis, Writing\u0026mdash;original draft; D.N.: Conceptualization, Methodology, Writing\u0026mdash;review and editing, Supervision. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eW. Gouveia, J. F. Tiago, S. R. Martins, M. Meireles, M. Carolino, S. Almeida, T. V. Almeida and M. E. Araujo, \"Toxicity of ionic liquids prepared from biomaterials,\" \u003cem\u003eChemosphere\u003c/em\u003e, vol. 104, pp. 51\u0026ndash;56, 2014.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eC. Agatemor,. K. N. Ibsen, E. E. Tanner and M. Samir, \"Ionic liquids for addressing unmet needs in healthcare,\" \u003cem\u003eBioengineering\u003c/em\u003e \u0026amp; \u003cem\u003eTranslational Medicine\u003c/em\u003e, vol. 3, pp. 7\u0026ndash;25, 2018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK. S. Egorova, E. G. Gordeev and V. P. 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FAIRBANKS, \"Antimicrobial therapy in Otolaryngology\u0026ndash;Head \u0026amp; Neck Surgery,\" \u003cem\u003eAmerican Academy of Otolaryngology\u0026ndash;Head\u003c/em\u003e \u0026amp; \u003cem\u003eNeck Surgery\u003c/em\u003e, vol. 13th ed, 2007.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme ","content":"\u003cp\u003eSchemes 1 is available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"linear polymers, ampicillin, cloxacillin, ionic liquid, choline, drug delivery system","lastPublishedDoi":"10.21203/rs.3.rs-4580822/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4580822/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe novel single and dual drug delivery systems (DDS) were designed by controlled polymerization of pharmaceutically functionalized choline-based monomers, i.e. [2-(methacryloyloxy)ethyl]trimethylammonium with counterions of cloxacillin (TMAMA/CLX), and ampicillin (TMAMA/AMP), providing the properties of antibiotics. This strategy was convenient to attain the well-defined linear copolymers with 38–93 mol. % of TMAMA contents, which were regulated by the initial ratio of TMAMA to methyl methacrylate comonomer. The compositions of polymers were controlled by the total monomer conversion (40–75%) resulting in a variable degree of polymerization (DP\u003csub\u003en\u003c/sub\u003e = 160–300) and contents of pharmaceutical anions (CLX¯ 51–80% and AMP¯ 78–87%). In aqueous solution, particles of the polymer achieved nanoscale sizes, measuring between 274–380 nm for CLX¯ systems and 288–348 nm for CLX¯/AMP¯ systems. In vitro drug release, which was driven by the exchange reaction of the pharmaceutical to phosphate anions in PBS, imitating a physiological fluid, occurred in the range of 58–76% of CLX¯ (10.5–13.6 µg/mL) in the single systems, and 91–100% of CLX¯ (12.9–15.1 µg/mL) and 97–100% of AMP¯ (21.1–23.3 µg/mL) in the dual systems. In relation to the conventional systems delivering both antibiotics without polymer carrier, the studied choline-based polymer DDS, demonstrating effective content of drug(s) and their (co)release from the polymer carriers, seems to be a great alternative solution.\u003c/p\u003e","manuscriptTitle":"Linear copolymers based on cholinium functionalized with antibiotic anions for single– and dual–drug delivery systems","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-03 20:14:38","doi":"10.21203/rs.3.rs-4580822/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"867da9c2-8e09-4a5a-b1bf-ce8527cd4cb4","owner":[],"postedDate":"July 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":34012004,"name":"Physical sciences/Chemistry/Medicinal chemistry"},{"id":34012005,"name":"Physical sciences/Chemistry/Polymer chemistry"},{"id":34012006,"name":"Physical sciences/Chemistry/Synthesis"},{"id":34012007,"name":"Physical sciences/Chemistry"},{"id":34012008,"name":"Physical sciences/Materials science"}],"tags":[],"updatedAt":"2024-07-17T06:21:39+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-03 20:14:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4580822","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4580822","identity":"rs-4580822","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}