Antimicrobial activity and biocompatibility of slow-release hyaluronic acid-antibiotic conjugated particles

Antimicrobial activity and biocompatibility of slow-release hyaluronic acid-antibiotic conjugated particles

International Journal of Pharmaceutics 576 (2020) 119024 Contents lists available at ScienceDirect International Journal of Pharmaceutics journal ho...

2MB Sizes 0 Downloads 1 Views

International Journal of Pharmaceutics 576 (2020) 119024

Contents lists available at ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Antimicrobial activity and biocompatibility of slow-release hyaluronic acidantibiotic conjugated particles ⁎

Ze Zhanga, Selin S. Sunerb, Diane A. Blakec, Ramesh S. Ayyalaa,d, , Nurettin Sahinerb,d,

T



a

Department of Ophthalmology, School of Medicine, Tulane University, New Orleans 70112 LA, USA Department of Chemistry, Faculty of Science & Arts, and Nanoscience and Technology Research and Application Center (NANORAC), Canakkale Onsekiz Mart University, Terzioglu Campus, 17100 Canakkale, Turkey c Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, LA 70448, USA d Department of Ophthalmology, School of Medicine, University of South Florida, Eye Institute, 12901 Bruce B. Downs Blvd., Tampa, FL 33612, USA b

A R T I C LE I N FO

A B S T R A C T

Keywords: Hyaluronic acid microgel/nanogel Ophthalmic drug delivery Antibiotic release system

Here, the aim was to design and use a long-lasting antibiotic release system for prevention of postoperative infections in ophthalmic surgery. Ciprofloxacin and vancomycin-conjugated hyaluronic acid (HA) particles were prepared as drug carriers for sustained release of antibiotics. The antimicrobial effects of the released drugs were determined by disc-diffusion and macro-dilution tests at different times up to 2 weeks. Slow degradable HA particles were obtained with 35.2 wt% degradation within 21 days. The drug loading amount was increased by employing two sequential chemical linking (conjugation, 2C) and one physical absorption loading (A) procedures (2C + A processes) from 148 ± 8 to 355 ± 11 mg/g HA particles for vancomycin. The amounts of vancomycin and ciprofloxacin that were released linearly was estimated as 64.35 ± 7.35 and 25.00 ± 0.68 mg/g, respectively, from drug-conjugated HA particles in 100 h. Antimicrobial studies revealed that antibiotic-conjugated HA particles could inhibit the growth of microorganisms from 1 h to 1 week. The MBC values were measured as 0.25, 4.0, and 0.25 mg/mL against Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis, respectively, after 72 h incubation time. Cytotoxicity studies showed no difference between fibroblast growth or corneal thickness after 5 days with or without HA-antibiotic particles. The drug release studies and antimicrobial activity of antibiotic-loaded HA particles with time against various bacteria further revealed that HA particles are very effective in preventing bacterial infections. Likewise, cytotoxicity studies suggest that these particles pose no toxicity to eukaryotic cells, including corneal endothelium.

1. Introduction Hyaluronic acid (HA) is a well-known natural polymeric disaccharide containing N-acetyl glucosamine and glucuronic acid groups with abundant –OH and –COOH functionality on main repeating units (Vanhee et al., 2017). It is a favored material among natural biopolymers in ophthalmic applications as it is native and the main component of many mammalian tissues: including extracellular matrices, synovial fluid in joints, dermis of the skin, and vitreous humor (Fraser et al., 1997; Galvin et al., 2016; Robert et al., 2010). HA-based materials have long been studied for ophthalmic treatments as an ocular carrier material because of HA’s inert nature and intraocular safety, tolerability (Galvin et al., 2016; Martens et al., 2015), stimulation of corneal and conjunctival cell proliferation (Artini et al., 2018), and the existence in

the retina and nerve fiber layer (Galvin et al., 2016; Hollyfield et al., 1998) Along with these properties it also has biodegradable, biocompatible, non-immunogenic, soft and viscoelastic features (Fallacara et al., 2018; Salwowska et al., 2016). Therefore, HA-based materials in different formulations such as hydrogel (Colter et al., 2018; Desai et al., 2018; Vil’danova et al., 2014), nanocarriers (Horvát et al., 2015), microneedles (Galvin et al., 2016), and niosomes (Zeng et al., 2016) have been used in ophthalmic drug delivery for the treatment of glaucoma (Wang et al., 2018), to inhibit angiogenesis (Galvin et al., 2016), for treatment of corneal conditions and injuries (Colter et al., 2018), and retinal gene therapy (Martens et al., 2015). HA-based materials can be further improved upon to increase ocular drug bioavailability by direct interaction with the precorneal mucin layer via inherently mucoadhesive effects. These can increase the residence time of the drug on the



Corresponding authors at: Department of Ophthalmology, School of Medicine, University of South Florida, Eye Institute, 12901 Bruce B. Downs Blvd., Tampa, FL 33612, USA (R.S. Ayyala). Department of Chemistry, Faculty of Science & Arts, and Nanoscience and Technology Research and Application Center (NANORAC), Canakkale Onsekiz Mart University, Terzioglu Campus, 17100 Canakkale, Turkey (N. Sahiner). E-mail addresses: [email protected] (R.S. Ayyala), [email protected], [email protected] (N. Sahiner). https://doi.org/10.1016/j.ijpharm.2020.119024 Received 23 October 2019; Received in revised form 20 December 2019; Accepted 6 January 2020 Available online 09 January 2020 0378-5173/ © 2020 Elsevier B.V. All rights reserved.

International Journal of Pharmaceutics 576 (2020) 119024

Z. Zhang, et al.

hydrochloride (Mustafa Nevzat Pharmaceuticals, MN Pharma, Istanbul, Turkey) were purchased and used as received. Pseudomonas aeruginosa ATCC 10145, Staphylococcus aureus ATCC 6538, and Bacillus subtilis ATCC 6633 were obtained from the Microbiology Department of School of Medicine at Canakkale Onsekiz Mart University. Nutrient agar (Microbiology grade) and nutrient broth (Microbiology grade) were purchased from Merck.

ocular surface, decrease drug loss, and improve bioavailability (Zeng et al., 2016). A major source of morbidity associated with ophthalmic surgery is the risk of post-operative infections. The rate of infections can be minimized by preoperative, intraoperative, perioperative, and postoperative prophylaxis measures (Epstein, 2011). One of the major challenges for postoperative infection prevention after ophthalmic surgery is the difficulty in administering topical medications to the patients. Studies have shown that up to 50% of prescribed drops are incorrectly instilled or not used at all (Stone et al., 2009). The ophthalmology community has attempted to find alternative solutions, including intracameral, transzonular, and intravitreal antibiotics. Each method has its limitations, including the short duration of action of the drug after intracameral injection, and higher risk of posterior infections and other complications associated with transzonular or intravitreal injections. Antibiotic carrying microparticle systems derived from different natural sources such as chitosan (De Campos et al., 2001; Siafakaa et al., 2015), cyclodextrin (Loftssona and Stefánsson, 2017), gelatin (Mahor et al., 2016), alginate (Costa et al., 2015), carboxymethylcellulose, chondroitin sulfate, and gellan gum have been reported for ocular delivery (Imperiale et al. 2018). These materials have many advantages similar to HA microparticles in ophthalmic antibiotic delivery because of their mucoadhesive properties, improving the precorneal residence time enabling drug bioavailability, and viscoelastic ability to protect corneal endothelium, and so on (Bonferoni et al., 2007a; Bonferoni et al., 2007b; Dubald et al., 2018). However, the longterm and sustainable drug release from these templates is inadequate for prolonging in vivo efficacy. Therefore, in this study, we aimed to create a long-lasting sustained release formulation of antibiotics from antibiotic-conjugated HA microparticles that can predictably release the desired medication at a certain rate over a desired period of time. This would allow coverage for infection prophylaxis over a longer time period, while eliminating the need for patients to administer postoperative drops, simplifying regimens and optimizing outcomes. Therefore, HA microparticles were synthesized with three different crosslinker ratios and degradable HA particles were loaded with two different antibiotics, namely, vancomycin and ciprofloxacin, with 2 methods. One method is a chemical conjugation process where the drug molecules are chemically linked to the polymeric network, and the second method is loading the drug molecules into HA particles by physical absorption by soaking HA particles in the drug solution. In order to increase the drug loading capacity, drugs can be loaded multiple times by conjugation e.g., after two times chemical conjugation, then physical absorption one or more times. The release profiles of these drugs from drug-conjugated HA particles were determined at pH 7.5 in balanced salt solution (BSS) at 37 °C, and the antimicrobial effects were evaluated against Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis at different contact times. In addition, cytotoxicity on fibroblasts and the effects on corneal thickness of these drug-conjugated HA particles were also investigated.

2.2. Synthesis of HA particles HA particles were synthesized in according to the procedures reported previously, with some modifications (Sahiner et al., 2017; 2019). Briefly, linear HA was dissolved in 0.2 M NaOH at a concentration of 50 mg/L. Then, 1.08 mL of this HA solution was transferred to 30 mL of 0.2 M AOT solution in isooctane. The mixture was immediately vortexed until a clear suspension was obtained. Three different mole ratios of the crosslinker GDE at 50, 100 and 200 mol% relative to the HA repeating unit were subsequently added to the mixture and then vortexed again to disperse the GDE. The reaction was allowed to proceed for 1 h at ambient temperature with vigorous stirring at 1200 rpm. Then, the obtained particles were precipitated in excess acetone and purified by centrifugation at 10,000 rotations per minute (rpm) for 10 min (min) at 20 °C. This was followed by removal of the supernatant solution and re-dispersal with acetone and re-centrifugation at least three times. Finally, the prepared HA particles were dried with a heat gun at cold adjustment and low blowing speed and kept in a closed container for further use. 2.3. Characterization of HA particles The prepared HA particles were characterized in terms of shape and size via optic microscope (BX53, Olympus) and scanning electron microscope (SEM, Jeol JSM-5600 LV). The size distributions and zeta potential values of HA particles were measured by using Dynamic Light Scattering (DLS, 90 plus, Brookhaven Inst. Corp.) and zeta potential analyzer (ZetaPals, BIC). In DLS and zeta potential measurements, HA particles at 2 mg/mL concentration were suspended in BSS and the measurements were carried out. The hydrolytic degradation of HA particles with three different crosslinker ratios were investigated at pH 7.4 in 0.01 M phosphate buffer solution (PBS) at 37 °C according to previously reported procedure (Sahiner et al., 2019). In the hydrolytic degradation experiments, 50 mg HA particles was dispersed in 50 mL PBS solution at 37 °C in a water bath shaker, and the degradation amounts were measured by high performance liquid chromatography (HPLC, Thermo Ultimate 3000) with a refractive index (RI) detector and 5 µm, 300 mm × 7.8 mm size of rezex RNM-Carbohydrate Na+ column (phenomenex). The amount of degraded HA was determined using the elution solution from the degradation flask with time against the previously prepared HA calibration curve in PBS at about 4 min retention time. The HPLC conditions were adjusted at 0.8 mL/min flow rate of 5 mM of sulfuric acid solution as a mobile phase, and 35 °C temperature of column and RI detector with 20 µL of injection volume and for 10 min run time. The weight losses (%) of HA particles were evaluated with time constructed to estimate the degrading of amount HA with time. All the experiments were repeated three times.

2. Materials and methods 2.1. Materials Sodium hyaluronate (95%, Acros Organics) as natural biopolymer, glycerol diglycidyl ether (GDE, technical grade, Aldrich) as crosslinker, sodium bis(2-ethylhexyl) sulfosuccinate (AOT, 98%, Sigma-Aldrich) as a surfactant, 2,2,4-trimethylpentane (isooctane, ACS, Reag. Ph Eur, 99.5%) as solvent, and 1,1′-carbonyldiimidazole (≥97.0%, SigmaAldrich) as a coupling agent were used as received. All the solvents; acetone (puriss., ≥99%, Sigma-Aldrich), ethanol (99%, Birkim) and dimethyl sulfoxide (puriss., ACS reagent, ≥99.5%, Sigma-Aldrich) were of the highest purity available. Ultra-pure distilled water 18.2 MΩ cm (Millipore-Direct Q UV3) was used throughout the studies. Ciprofloxacin (≥98%, HPLC grade, Sigma-Aldrich) and vancomycin

2.4. Drug loading experiments Ciprofloxacin and vancomycin drugs were loaded into HA particles by two methods; 1) chemical conjugation (C) that was repeated twice (2C), and 2) a physical absorption (A) process in accordance with the previously published method (Sahiner et al., 2019; Suner et al., 2019a; Zhang et al., 2014). Briefly, 0.532 g of ciprofloxacin or 0.42 g vancomycin was dissolved in 10 mL of DMSO solution. Then, 0.286 g of 1,1′carbonyldiimidazole was added to these drug solutions and stirred at 2

International Journal of Pharmaceutics 576 (2020) 119024

Z. Zhang, et al.

2.6.1. Disc diffusion Antimicrobial activity of the released drugs was tested on Pseudomonas aeruginosa (PA) and methicillin-resistant Staphylococcus aureus (MRSA) cultures and compared to standard concentrations of antibiotics using filter disks on Mueller Hinton plates for ciprofloxacinand vancomycin-conjugated HA particles, respectively. The released antibiotics or controls were added onto sterile filter discs (created using a 5 mm standard hole punch on filter paper, autoclaved). One colony of each bacteria was selected (PA or MRSA) and placed into a bacterial culture tube, incubated in 5 mL of LB broth and placed on a 37 °C shaker for approximately 4–6 h. Of the incubated bacteria, 500 µL was then added to the Mueller-Hinton Plates (which were acclimated to room temperature for at least 2 h) and a plastic disposable spreader was used to carefully spread the bacterial broth onto the plates and allowed to settle for approximately 5–10 min. The filter discs were then placed on top of the bacterial plate and the plates were inverted and placed in the 37 °C incubator overnight. After 24 h, the plates were removed, and the zones of inhibition measured and photographed.

room temperature for 1 h. Subsequently, 0.84 g HA particles was added to this mixture and the reaction was allowed to continue for 24 h at 80 °C under stirring. Then, these one-time drug-conjugated (1C) HA particles were washed with DMSO by centrifugation at 10,000 rpm. Then, the 1C HA particles were conjugated with ciprofloxacin or vancomycin again by employing the same procedure described above and washed with DMSO again to prepare two-times drug-conjugated (2C) HA particles. These 2C HA particles were put into 20 mL of DMSO solution containing 0.1 g ciprofloxacin or vancomycin drug for physical absorption (A) and stirred at room temperature for 12 h for the physical drug loading process. The two-times drug-conjugated, and one-time drug-adsorbed (2C + A) HA particles were washed with DMSO once, washed with acetone once and dried with a heat gun at cold adjustment with low blowing speed for further use. The drug loading amounts and loading capacity were determined from the absorbance of the drug solution before and after the loading process by using UV–VIS spectroscopy at 270 nm and 282 nm for ciprofloxacin and vancomycin, respectively, against the previously created corresponding drug calibration curves prepared in DMSO. These are calculated from the following equations:

mg ⎞ Drug loading amount ⎛⎜ ⎟ ⎝ g ⎠ Total drug amount − Remaining drug amount = Weight of particles

(1)

Drug loading capacity(%) Total drug amount − Remaining drug amount = × 100 Total drug amount

(2)

2.6.2. Macro dilution Antimicrobial properties of bare and vancomycin-conjugated (2C + A) HA particles against Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis (BS) bacterial strains were investigated at different incubation times; 24 h, 48 h, and 72 h. The HA particles were sterilized by photo irradiation at 420 nm for 2 min. Five concentrations ranging from 2.5 to 40 mg of bare and drug-loaded HA particles were placed into 10 mL of nutrient broth and 100 µL of bacterial culture, which was adjusted to 1 × 108 colony forming unit (CFU)/mL with McFarland 0.5 standard, was added to the broth medium. The tubes were incubated at 35 °C for 24, 48, and 72 h incubation times in a water bath shaker. The minimum inhibition concentration (MIC) values were determined as the lowest concentration of transparent tubes which contains no visible growth. Then, 100 µL of the transparent tubes were inoculated on nutrient agar to detect the surviving bacterial counts and minimum bactericidal concentration (MBC) values were determined as the minimum concentration that kills 99% of bacteria in medium.

2.5. Drug release experiments For the drug release experiments, the previously reported procedure was adopted with some modifications (Sahiner et al., 2019; Suner et al., 2019a). In the drug release studies, 50 mg of drug-conjugated HA particles were dispersed in 1 mL of balanced salt solution (BSS) at pH 7.5 and transferred to a dialysis membrane. The membrane containing drug-loaded HA particles (MW cut off 12kDA) was placed into 30 mL of BSS solution (pH 7.5) at 37 °C in a shaker bath. The drug releasing medium, BSS solution, was then sampled and evaluated by UV–Vis spectrometer at 270 nm and 280 nm for ciprofloxacin and vancomycin against the previously determined corresponding drug calibration curves prepared in BBS, and the released amounts of drug were calculated. Each 24 h, the BSS solution containing the released drug was discarded and 30 mL of fresh BSS solution was placed into the release medium and the determination of drug release amount continued using the UV–Vis spectrometer again. The analysis was repeated three times, and the values are reported as the average values with standard deviations. For the drug release kinetic from HA particles, Korsmeyer-Peppas kinetic model was adopted (Korsmeyer et al., 1983). The release kinetic data obtained from plotted as log cumulative % drug release versus log time (h) according to the following Korsmeyer-Peppas equation:

Mt / M∞ = kt n

2.7. Cytotoxicity experiments The cytotoxicity test of bare and drug-loaded HA particles were investigated as previously described (Ponnusamy et al., 2014). In the cytotoxicity studies, 1 × 105 smooth muscle cells were plated into each well of a 12-well plate and allowed to settle for 1 h in 2.5 mL of complete culture media (DMEM + 10% fetal bovine serum + antibiotics + amino acids). The cells were allowed to grow to 70–80% confluence. Meanwhile, various concentrations of drug-loaded HA particles, bare HA particles, and antibiotics were suspended in complete media and added to the wells. The cells were incubated at 37 °C 95% CO2 for 5 days. Following the incubation period, the culture media was removed and cells were fixed in glutaraldehyde then stained with toluidine blue and resuspended in sodium dodecyl sulfate (SDS). A cell counter was then used to quantify the absorbance of the stain to quantify and compare the number of cells in each group. The plates were photographed, resuspended and monitored by UV–Vis spectrophotometer to measure the light absorbance of the cell density.

(3) 2.8. Corneal thickness

where Mt/M∞ is the fractional of drug released at time t, k and n are the release rate constant related to drug/polymer interaction and the diffusional exponent.

Corneal toxicity of the polymers was examined by incubating human donor corneas in the presence or absence of polymers as evidenced by increasing corneal thickness. Human donor corneas were obtained from the Southern eye bank (New Orleans, LA) and suspected in optisol solution. Pachymetry was measured using a standard corneal pachymeter. Drug-conjugated HA particles weighing 30 and 90 µg were then added to the cornea in optisol and allowed to incubate for 5 days in 37 °C. After 5 days, the corneal pachymetry was again measured and

2.6. Antimicrobial experiments Antimicrobial properties of bare and drug loaded HA particles were determined using disc diffusion and macro dilution tests in accord with the literature (Suner et al., 2019b). 3

International Journal of Pharmaceutics 576 (2020) 119024

Z. Zhang, et al.

Fig. 1. (a) Optic microscope images of dry and swollen HA particles crosslinked with GDE at 50% mole ratio of crosslinker, (b) the corresponding SEM images of dry HA particles, and (c) Hydrolytic degradation of HA particles crosslinked with GDE at three different crosslinker ratios, 50, 100, and 200% mole ratio of HA repeating unit.

3.1. Synthesis and characterization

the endothelial cells were observed under high magnification specular microscopy.

The optic microscope and SEM images of the HA particles crosslinked with GDE at 50% mole ratio of HA repeating unit are shown in Fig. 1a and 1b. As seen in Fig. 1a, the dry HA particles ranged from 0.5 to 15 µm in size with (also confirmed by SEM, in Fig. 1c) spherical shapes and smooth surfaces. Upon immersing HA particles in DI water, HA particles swell in size, increasing by a few tens of micrometers. The degradation behavior of drug carrier vehicles is one of the important parameters to design of sustainable drug delivery systems. Types of crosslinker bounding groups such as ester or ether linkages (Yui et al., 1992) as well as crosslinker degree are significantly predictive factors of the crosslinker degradations (Sahiner et al., 2019). Therefore, three dimensional polymeric networks of HA particles as an antibiotic carrier material were prepared by glycerol diglycidyl ether (GDE) crosslinker at different mole degree which containing two degradable epoxy groups

3. Results and discussion Patient adherence to topical ophthalmic medications is a major barrier to quality of life and postoperative outcome in ophthalmic practice. Both clinical outcome and patient quality of life would be greatly improved by a sustained release antibiotic that could be administered at time of surgery and eliminate the need for topical medications, greatly simplifying the postoperative course for the patient (Ayyala et al., 2016). HA particles were utilized as a drug carrier in many biomedical applications because of their innate soft, biocompatible, degradable, and non-toxic properties (Sahiner et al., 2012)

4

International Journal of Pharmaceutics 576 (2020) 119024

Z. Zhang, et al.

were observed after drug conjugation into HA particles. In addition, pH values of the BSS solutions containing particles did not change with the types of drug and/or chemical conjugation number. But, the zeta potential values of the HA particles decreased from –33.1 ± 1.4 mV for bare HA particles to −6.8 ± 3.6 mV and −9.1 ± 2.8 mV values for 2C + A vancomycin- and ciprofloxacin-loaded HA particles, respectively. As reported, the zeta potential values of negatively-charged materials were significantly increased after loading vancomycin (Ritsema et al., 2018) or ciprofloxacin (Cheng et al., 2018) onto HA particles because of the existence of positively-charged functional groups (amines) in the molecular structures of the drug molecules. These results confirm that vancomycin or ciprofloxacin drugs can be successfully linked into HA particles. The drug loading amount (mg/g), loading capacity (%), release amount (mg/g), and release capacity % for one time conjugated (1C), two times conjugated (2C) and drug absorption into 2C HA particles as 2C + A particles are given in Table 2 for 200 h release time. From these results, vancomycin loading amounts for 1C, 2C, and 2C + A HA particles were determined as 148 ± 8, 282 ± 6, and 355 ± 11 mg/g. In addition, vancomycin loading capacity % of HA particles were about the same, changing between 29.6 and 32.2% for all steps, whereas the total drug loading amount of HA particles can be significantly increased by multiple chemical conjugation and physical absorption processes. Drug loading amount commonly depend on the used drug loading process, times (Sagbas and Sahiner, 2018) and the chemical structure of drug and carriers (Rizvi and Saleh, 2018). Therefore, one-time conjugation of the drug may not completely make use of all the functional groups on HA particles for chemical linking or physical absorption into HA particles. Therefore, several chemical conjugations of drugs and physical absorption processes allow loading of the desired amount of drug into HA particles. In this study, we were able to control the amount of drug loaded into HA particles and thereby affect the rate of release by employing multiple drug conjugations and absorption techniques for the HA particle network. Among the other drug loading processes, drug conjugation or the chemical linking process into the HA particle network, has more advantages as it allows for slow release kinetics and improves the efficacy of the drug delivery system along with higher and tunable drug loading capacity, and sustainable and long-term drug release rates (Suner et al., 2019a; Zhang et al., 2014). Vancomycin and ciprofloxacin were used in our drug delivery applications to determine the potential usability of HA particles as antimicrobial drug carrier material for the inhibition of infection. These drugs were conjugated into the HA particle network by using both loading procedures in accordance with the previously reported process (Sahiner et al., 2019). According to this process, CDI was utilized as a coupling agent between carboxylic acid groups of the drug and hydroxyl groups of HA particles by esterification reactions that are cleavable in aquatic milieu (Elvira et al., 2005). Earlier, degradable and porous HA particles crosslinked with divinylsulfone were conjugated with vancomycin only once (1C) in the literature (Sahiner et al., 2019). In this study degradable HA particles crosslinked with GDE were conjugated with this drug two times and then soaked in drug solution to physically load the drug by absorption (2C + A) to increase the loading capacity of the particles. The drug release profiles (mg/g) and (%) of vancomycin-loaded particles at pH 7.5 in balanced salt solution (BSS) at 37 °C are demonstrated in Fig. 2a and b. As shown in Fig. 2, vancomycin was gradually released for about 60 h, with low release rates persisting up to 200 h. Moreover, about 16–18 percentage of the loaded vancomycin was released as 24.21 ± 1.81, 45.31 ± 2.43, and 64.35 ± 7.35 mg/ g from 1C, 2C, and 2C + A HA particles in 200 h as seen in Fig. 2b. These results confirm that vancomycin loading and release was almost three-fold increased by multiple drug conjugation and drug absorption processes in comparison to 1C HA particles. Korsmeyer-Peppas model was applied for the antibiotic release kinetic and the corresponding n and R2 values for all HA based carrier

for slightly and prolonger drug release. The hydrolytic degradability of these HA particles crosslinked with three different crosslinker ratios, 50, 100, and 200% mole ratio, were followed for up to 35 days, and the results are illustrated in Fig. 1c. No significant hydrolytic degradation was observed for 200% mole ratio of crosslinked HA particles, whereas there were some degradations for 50 and 100% mole ratio of crosslinked HA particles, e.g., 35.2% and 21.5% weight losses, respectively, within 21 days. The degradation of HA particles gradually increased with time and leveled off after two weeks. Degradable HA hydrogels bounding by epoxy group containing crosslinker are reported an earlier study (Yui et al., 1992). These studies supported that the epoxy group containing crosslinker in the HA microparticle network could be degraded by the swelling and ionic effects of the balanced salt solution depending on the amount of crosslinker used. These results indicate that crosslinker ratio has a significant effect on the degradation of HA particles with an inverse relationship between amount of crosslinker used and hydrolytic degradation amount 3.2. Drug loading and release To avoid the side effects of antibiotics, improve their bioavailability, and increase the loading and release efficiency in ocular system, microparticle systems such as the ones derived from HA, that has a wide range of usage (Galvin et al., 2016; Martens et al., 2015) in ocular system were used as a carrier system for vancomycin and ciprofloxacin antibiotics. These model drugs were chemically linked into HA particles by chemical conjugation (C) and physical absorption (A) methods to enhance and control the drug loading and release efficiencies. In the chemical conjugation process, drugs (vancomycin or ciprofloxacin) reacted 1,1′-carbonyldiimidazole (CDI) coupling agent in DMSO for 1 h and CDI was the removed from the drugs by reacting with the hydroxyl groups of HA particles as demonstrated in Supporting Fig. 1. This coupling agent is well known and widely used material in drug conjugation process because of its’ readily biodegradable ester bond generation capability between the drug and carrier materials (Elvira et al., 2005). In this process, HA particles were added into this drug medium and reacted for 12 h at 80 °C and vancomycin or ciprofloxacin drugs were conjugated with HA particles by esterification reaction between carboxylic acid of the drugs and hydroxyl groups of HA particles in accordance with the previously published method (Sahiner et al., 2019; Sagbas and Sahiner, 2018; Suner et al., 2019a; Zhang et al., 2014). Onetime chemical linking of drugs to HA particles is called 1C, and the use of 1C HA particles for chemically linking the drug was called 2nd time 2C, and physically placing HA particles (non-conjugated HA or 1C or 2C) into drug solutions is called physical absorption. Therefore, to increase the drug loading amounts and obtained the desired release amounts from HA particles, the methods of 1C, 2C and A and/or their combinations were employed. The particle sizes, pH values and zeta potential values of bare and drug-loaded HA particles in BSS at 2 mg/ mL concentration are given in Table 1. According to the results, bare HA particles had size of 1913 ± 269 nm via DLS measurement and no significant differences Table 1 Particle sizes, pH values and zeta potential values of bare HA particles, 1 times vancomycin conjugated (1C), 2 times vancomycin conjugated (2C) and physical drug adsorbed 2 times vancomycin- and ciprofloxacin-conjugated (2C + A) HA particles in pH 7.4 balanced salt solution. HA particles

Particle sizes (nm)

pH

Zeta Potentials (mV)

Bare 1C Vancomycin 2C Vancomycin 2C + A Vancomycin 2C + A Ciprofloxacin

1913 1887 1904 1911 1965

7.5 7.4 7.3 7.3 7.1

–33.1 ± 1.4 −17.8 ± 3.6 −10.4 ± 2.0 −6.8 ± 3.6 −9.1 ± 2.8

± ± ± ± ±

269 260 195 216 204

5

International Journal of Pharmaceutics 576 (2020) 119024

Z. Zhang, et al.

Table 2 Vancomycin and ciprofloxacin loading amounts (mg/g), loading capacity (%), release amount (mg/g), and release capacity (%), and release kinetic via KorsmeyerPeppas model for one time conjugated (1C), two times conjugated (2C) and physically drug absorption into 2C HA particles as 2C + A. HA particles

1C 2C 2C 2C

Vancomycin Vancomycin + A Vancomycin + A Ciprofloxacin

Loading amount (mg/g)

148 282 355 204

± ± ± ±

8 6 11 9

Loading capacity (%)

29.6 28.2 32.2 14.9

± ± ± ±

1.6 0.6 3.1 0.6

Releasing amount (mg/g)

24.21 45.31 64.35 25.00

± ± ± ±

1.81 2.43 7.35 0.68

Releasing capacity (%)

16.35 16.06 18.12 12.25

± ± ± ±

1.22 0.86 2.07 0.33

Release kinetic by Korsmeyer-Peppas model n

R2

0.19 0.32 0.29 0.07

0.89 0.95 0.96 0.90

that of vancomycin, (25.00 ± 0.68 mg/g) with about 12% release capacity from 2C + A loaded HA particles. Therefore, depending of the types of antibiotic, drug loading methods (conjugation or absorption), and the amount of crosslinker used during HA particle synthesis, the release capacity of the drugs can be adjusted to fit the desired rate of release. Many researches were reported the drug carriers for ocular therapy using natural polymeric based micro/nanoparticles (De Campos et al., 2001; Costa et al., 2015; Imperiale et al. 2018; Mahor et al., 2016). These drug carrier materials derived from chitosan (De Campos et al., 2001), gelatin (Mahor et al., 2016), and alginate (Costa et al., 2015) were reported to release the loaded drug in a relatively short time e.g., 24 h, 12 h, and 4 h, respectively. However, the antibiotic careering HA particles here were shown the delivery of of antibiotic for about 100–200 h because of their higher and versatile loading capacity by employing several conjugation and absorption processes. Therefore, amounts the natural polymeric carriers, HA microparticles

systems were reported in Table 2. The value of n is used to characterize drug release mechanisms from HA microparticles in accord with literature (Lungan et al., 2015). The values, n ≤ 0.45 implies the controllable drug release via Fickian diffusion, 0.45 < n < 0.89 values indicate the anomalous or non-Fickian transport, and n > 0.89 values represents the super Case II transport and zero order release kinetics (Dash et al., 2010). The n values of vancomycin loaded 1C, 2C, and 2C + A HA particles were found as 0.19, 0.32, and 0.29 with relatively high R2 values indicating the antibiotic loaded HA particles release occurs by Fickian diffusion. Moreover, another antibiotic drug, ciprofloxacin, was also loaded by 2C + A processes and the ciprofloxacin loading amount was measured as 204 ± 9 mg/g with 14.9 ± 0.6% loading capacity as reported in Table 2. The ciprofloxacin and vancomycin release profiles (mg/g) and (%) from 2C + A HA particles are illustrated in Fig. 2c and d. The released amount of ciprofloxacin was about 2.5-fold less than

Fig. 2. Vancomycin release profiles; (a) mg/g drug release and (b) percentage drug release from one-time (1C), and two-times (2C) drug conjugated, and physical drug adsorption of 2-times conjugated HA particles, 2C + A. Ciprofloxacin and Vancomycin release profiles; (c) mg/g drug release and (d) percentage drug release from ciprofloxacin adsorbed 2C + A HA particles at pH 7.5 in balanced salt solution at 37 °C. 6

International Journal of Pharmaceutics 576 (2020) 119024

Z. Zhang, et al.

Fig. 3. (a) Antimicrobial activity and (b) zone inhibition of ciprofloxacin-loaded 2C + A HA particles against Pseudomonas aeruginosa.

sufficient inhibition of PA and MRSA growth, respectively, at every time point of collection between 1 h and 7 days (zone of inhibition up to 15 mm). After 1 week, the rate of drug release appeared to be too low for inhibition of bacterial growth. The control studies show that only 10 µg of vancomycin on a filter disc was sufficient to inhibit MRSA growth and provide a zone of inhibition of 8 mm. It was reported that the minimum inhibitory concentration for ciprofloxacin is 0.5 µg/mL and for vancomycin is 1 µg/ mL (EUCAST, 2003). The amount of antibiotic released using HA particles was far in excess of the MIC value given that the volume of the human anterior chamber is only 250 µL. In addition, to simulate the constant out flow of aqueous humor and thus gradual decline in drug concentration in the eye, we removed 25% of the supernatant with the drug release collections each time we collected the sample and replaced with BSS, thus removing a significant amount of the drug each time,

can be assumed the most promising material for ophthalmological applications with tunable loading capability rendering sustainable and long-term antibiotic release kinetics. 3.3. Antimicrobial activity Antimicrobial activity of the drug released from 2C + A ciprofloxacin- and vancomycin-loaded HA particles was tested by the disc diffusion method on PA and MRSA cultures and compared to standard concentrations of ciprofloxacin and vancomycin, respectively, using filter disks on Mueller Hinton plates. The control studies showed that only 5 µg of ciprofloxacin, and 10 µg of vancomycin were sufficient to inhibit PA and MRSA growth, respectively, and provide a zone of inhibition that is well above the MIC values as shown in Figs. 3 and 4. The released ciprofloxacin and vancomycin were able to provide 7

International Journal of Pharmaceutics 576 (2020) 119024

Z. Zhang, et al.

Fig. 4. (a) Antimicrobial activity and (b) zone inhibition of vancomycin-loaded 2C + A HA particles against Methicillin-resistant Staphylococcus aureus.

caused by common bacteria. Antimicrobial activity of the 2C + A vancomycin-loaded HA particles were also determined by macro-dilution method against PA, SA, and BS bacteria strains at different incubation times of 24 h, 48 h, and 72 h and the results are given in Table 3. As stated earlier, the minimum inhibitory concentration (MIC) of vancomycin is about 1 µg/mL (EUCAST, 2003) and MIC values of vancomycin-conjugated HA particles were found as 0.25 mg/mL. This is plausible as vancomycin-conjugated HA particles can release nearly 0.4–0.5 µg/mL vancomycin within 72 h. It was also confirmed that vancomycin and ciprofloxacin were gradually released from the drugconjugated HA particles with the antimicrobial studies, inhibiting the

allowing antimicrobial testing using the actual change in drug concentration and not a cumulative effect. Even so, 20 mg of ciprofloxacinconjugated HA particles and 30 mg of vancomycin-conjugated HA particles were sufficient and effective at preventing bacterial growth at every time point between 1 h and 7 days, with zones of inhibition up to 15 mm and 12 mm, respectively, as illustrated in Figs. 3 and 4. It was shown that a polyethylene glycol derived antibiotic carrier system can prevent postoperative ocular infection for at least 1 week via sustainable drug release capability (Kashiwabuchi et al., 2017). Our results revealed that drug-conjugated HA particles are promising materials for ophthalmic treatments as ocular antibiotic carrier material with their potent inhibition effects lasting up to 1 week against many infections

8

International Journal of Pharmaceutics 576 (2020) 119024

Z. Zhang, et al.

Table 3 MIC and MBC values of bare and vancomycin conjugated HA particles (2C + A) against Pseudomonas aeruginosa ATCC 10145, Staphylococcus aureus ATCC 6538, and Bacillus subtilis ATCC 6633 bacterial strains at different incubation times. Particles

Minimum inhibition concentration (MIC) (mg/mL) P. aeruginosa (gram −) 24 h

HA 2C + A HA Particles

HA 2C + A HA

48 h

S. aureus (gram +) 72 h

B. subtilis (gram +)

24 h

48 h

72 h

24 h

48 h

72 h

– – – 0.5 0.25 0.25 Minimum bactericidal concentration (MBC) (mg/mL)

– 2

– 2

– 2

– 0.25

– 0.25

– 0.25

P. aeruginosa (gram -)

S. aureus (gram + )

B. subtilis (gram + )

24 h

48 h

72 h

24 h

48 h

72 h

24 h

48 h

72 h

– 1

– 0.5

– 0.25

– 4

– 4

– 4

– 1

– 0.5

– 0.25

Control

30 mg

90 mg

0.25

(a) Abs (nm)

0.2 0.15 0.1 0.05 0 Control Control

30 mg

30 mg

90 mg

90 mg 0.25

(b) Abs (nm)

0.2 0.15 0.1 0.05 0 Control

30 mg

90 mg

Fig. 5. Cytotoxicity studies of (a) ciprofloxacin-conjugated HA particles and (b) vancomycin-conjugated HA particles.

cell proliferation over 5 days under normal culture conditions. The presence of HA particles slightly decreased cell division or proliferation rates depending on the particle concentration. However, these decreases were not significant in comparison to controls where cells were cultured in the absence of any additional particles as illustrated in Fig. 5. These types of sustained release mechanisms provide adequate drug release in a timely manner and prevent adverse effects on human tissues due to the biocompatible nature of HA.

microorganism growth in accordance with initially high and then gradual drug release capability to prevent reoccurring bacterial infections. These HA particles can afford great potential for antimicrobial activity in ocular delivery system making them promising materials in long term and sustainable antibiotic release kinetic e.g., up to 200 h in comparison with literature (De Campos et al., 2001; Costa et al., 2015; Mahor et al., 2016) where most of these drug carrying micro/nanoparticles systems prepared from chitosan, gelatin, and also alginate lost their antimicrobial ability at about 24 h.

3.5. Corneal thickness studies 3.4. Cytotoxicity studies The presence of antibiotic-loaded HA polymer had no effect on corneal thickness over a 5 day incubation period as illustrated in Fig. 6

The antibiotic-loaded HA particles showed no effect on fibroblast 9

International Journal of Pharmaceutics 576 (2020) 119024

Z. Zhang, et al.

patients with glaucoma and dry-eye syndrome: A double-blind randomized clinical trial. J. Phys.: Conf. Series 1073, 032075. Ayyala, D., Blake, D.A., John, V.T., Ayyala, R.S., 2016. Biomaterials and regenerative medicine in ophthalmology. In: Chirila, T.V., Harkin, D.G. (Eds.), 14 – A glaucoma drainage device incorporating a slow-release drug delivery system for the management of fibrosis. Woodhead Publishing Series in Biomaterials, pp. 346367. Bonferoni, M.C., Sandri, G., Chetoni, P., Rossi, S., Ferrari, F., Caramella, C., 2007a. Microparticle systems based on polymer-drug interaction for ocular delivery of ciprofloxacin II. Precorneal residence times. J. Drug Del. Sci. Tech. 17, 63–68. Bonferoni, M.C., Sandri, G., Gavini, E., Rossi, S., Ferrari, F., Caramella, C., 2007b. Microparticle systems based on polymer-drug interaction for ocular delivery of ciprofloxacin I. In vitro characterization. J. Drug Del. Sci. Tech. 17, 57–62. Cheng, R., Li, H., Liu, Z., Du, C., 2018. Halloysite nanotubes as an effective and recyclable adsorbent for removal of low-concentration antibiotics ciprofloxacin. Minerals 8, 387. Colter, J., Wırostko, B., Coats, B., 2018. Finite element design optimization of a hyaluronic acid-based hydrogel drug delivery device for improved retention. Ann. Biomed. Eng. 46, 211–221. Costa, J.R., Silva, N.C., Sarmento, B., Pintado, M., 2015. Potential chitosan-coated alginate nanoparticles for ocular delivery of daptomycin. Eur. J. Clin. Microbiol. Infect. Dis. Off. Publ. Eur. Soc. Clin. Microbiol. 34, 1255–1262. Dash, S., Murthy, P.N., Nath, L., Chowdhury, P., 2010. Kinetic modeling on drug release from controlled drug delivery systems. Acta Pol. Pharm. 67, 217–223. De Campos, A.M., Sánchez, A., Alonso, M.J., 2001. Chitosan nanoparticles: a new vehicle for the improvement of the delivery of drugs to the ocular surface. Application to cyclosporin A. Int. J. Pharm. 224, 159–168. Desai, A.R., Maulvi, F.A., Pandya, M.M., Ranch, K.M., Vyas, B.A., Shah, S.A., Shah, O., 2018. Co-delivery of timolol and hyaluronic acid from semi-circular ring-implanted contact lenses for the treatment of glaucoma: in vitro and in vivo evaluation. Biomater. Sci. 6, 1580–1591. Dubald, M., Bourgeois, S., Andrieu, V., Fessi, H., 2018. Ophthalmic drug delivery systems for antibiotherapy - a review. Pharmaceutics 10, 10. Elvira, C., Gallardo, A., Roman, J.S., Cifuentes, A., 2005. Covalent polymer-drug conjugates. Molecules 10, 114–125. Epstein, N.E., 2011. Preoperative, intraoperative, and postoperative measures to further reduce spinal infections. Surg. Neurol. Int. 2, 17. European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID). 2003. Determination of minimum inhibitory concentrations (MICs) of antibacterial agents by broth dilution. Clin. Microbiol. Infect. 9, 1–7. Fallacara, A., Baldini, E., Manfredini, S., Vertuani, S., 2018. Hyaluronic acid in the third millennium. Polymers 701, 1–36. Fraser, J.R., Laurent, T.C., Laurent, U.B., 1997. Hyaluronan: its nature, distribution, functions and turnover. J. Intern. Med. 242, 27–33. Galvin, O., Srivastava, A., Carroll, O., 2016. A sustained release formulation of novel quininib-hyaluronan microneedles inhibits angiogenesis and retinal vascular permeability in vivo. J. Control Release 233, 198–207. Hollyfield, J.G., Rayborn, M.E., Tammi, M., Tammi, R., 1998. Hyaluronan in the interphotoreceptor matrix of the eye: species differences in content, distribution, ligand binding and degradation. Exp. Eye Res. 66, 241–248. Horvát, G., Budai-Szucs, M., Berkó, S., Szabó-Rézész, P., Soós, J., Facskó, A., Maroda, M., Mori, M., Sandri, G., Benferoni, M.C., Caramella, C., Csányi, E., 2015. Comparative study of nanosized cross-linked sodium-, linear sodium and zinc-hyaluronate as potential ocular mucoadhesive drug delivery systems. Int. J. Pharm. 494, 321–328. Imperiale, J.C., Acosta, G.B., Sosnik, A., 2018. Polymer-based carriers for ophthalmic drug delivery. J. Control. Release 285, 106–141. Kashiwabuchi, F., Parikh, K.S., Omiadze, R., Zhang, S., Luo, L., Patel, H.V., Xu, Q., Ensign, L.M., Mao, H.-Q., Hanes, J., McDonnell, P.J., 2017. Development of absorbable, antibiotic- eluting sutures for ophthalmic surgery. Transl. Vis. Sci. Techn. 6, 1. Korsmeyer, R.W., Gurny, R., Doelker, E., Buri, P., Peppas, N.A., 1983. Mechanisms of solute release from porous hydrophilic polymers. Int. J. Pharm. 15, 25–35. Lungan, M.-A., Popa, M., Racovita, S., Hitruc, G., Doroftei, F., Desbieres, J., Vasiliu, S., 2015. Surface characterization and drug release from porous microparticles based onmethacrylic monomers and xanthan. Carbohdr. Polym. 125, 323–333. Loftssona, T., Stefánsson, E., 2017. Cyclodextrins and topical drug delivery to the anterior and posterior segments of the eye. Int. J. Pharm. 531, 413–423. Mahor, A., Prajapati, S.K., Verma, A., Gupta, R., Iyer, A.K., Kesharwani, P., 2016. Moxifloxacin loaded gelatin nanoparticles for ocular delivery: Formulation and invitro, in-vivo evaluation. J. Colloid Interface Sci. 483, 132–138. Martens, T.F., Remaut, K., Deschout, H., 2015. Coating nanocarriers with hyaluronic acid facilitates intravitreal drug delivery for retinal gene therapy. J. Control Release 202, 83–92. Ponnusamy, T., Yu, H., John, V.T., Ayyala, R.S., Blake, D.A., 2014. A novel antiproliferative drug coating for glaucoma drainage devices. J. Glaucoma 23, 526–534. Ritsema, J.A.S., Herschberg, E.M.A., Borgos, S.E., Løvmo, C., Schmid, R., te Welscher, Y.M., Storm, G., van Nostrum, C.F., 2018. Relationship between polarities of antibiotic and polymer matrix nanoparticle formulations based on aliphatic polyesters. Int. J. Pharm. 548, 730–739. Rizvi, S.A.A., Saleh, A.M., 2018. Applications of nanoparticle systems in drug delivery technology. Saudi Pharm. J. 26, 64–70. Robert, L., Robert, A.M., Renard, G., 2010. Biological effects of hyaluronan in connective tissues, eye, skin, venous wall. Role in aging. Pathol. Biol. 58, 187–198. Sagbas, S., Sahiner, N., 2018. Modifiable natural gum based microgel capcules as sustainable drug delivery systems. Carbohydr. Polym. 200, 128–136. Sahiner, N., Sagbas, S., Ayyala, R.S., 2019. Mesoporous, degradable hyaluronic acid microparticles for sustainable drug delivery application. Colloids Surf. B 177,

Fig. 6. Corneal thickness after treatment with antibiotic-polymer for 5 days.

compared to control group. Corneal thickness is an indicator of corneal endothelial health. There was no evidence of any difference between the control group or the HA polymer groups. We successfully created a slow-release HA linked antibiotic system that can potentially be used during intraocular surgery such as cataract surgery. This would simplify the postoperative regimen for patients while also minimizing postoperative infection by providing a predictable drug release profile for a set duration. 4. Conclusions Biocompatible and degradable HA microparticles with tunable degradability and high antibiotic loading capability offer great advantages over current drug administration techniques. The amounts of drugs loaded and the degradation rate of HA particles can be fine-tuned to obtain sustainable and long-term release profiles from these drug-conjugated HA particles that can inhibit the growth of bacteria for up to one week, as needed for postoperative infection prevention following ophthalmic surgery. Therefore, the use of HA particles to generate antibacterial materials via the slow release concept provides a new avenue for postoperative ophthalmic applications and for applications about various eye-related infectious diseases. Our future studies will involve using in vivo animal models to compare the prevention and treatment of post-operative infections in ophthalmic surgery. CRediT authorship contribution statement Ze Zhang: Methodology, Investigation. Selin S. Suner: Investigation, Writing - original draft. Diane A. Blake: Resources, Supervision. Ramesh S. Ayyala: Conceptualization, Resources, Supervision. Nurettin Sahiner: Methodology, Resources, Conceptualization, Supervision, Writing - original draft, Writing - review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ijpharm.2020.119024. References Artini, W., Hasudungan, V.C., Susiyanti, M., Prihartono, J., Gondhowiardjo, T.D., 2018. Comparison of the goblet cells’ density and the quality of tears in treatment with sodium hyaluronate 0.1% benzalkonium chloride preservative-free eye drops for

10

International Journal of Pharmaceutics 576 (2020) 119024

Z. Zhang, et al.

nanotubes with polyethyleneimine and various ionic liquid forms with antimicrobial activity. J. Appl. Polym. Sci. 137, 48352. Vanhee, C., Desmedt, B., Baudewyns, S., Kamugisha, A., 2017. Characterization of suspected dermal fillers containing hyaluronic acid. Anal. Methods 9, 4175–4183. Vil’danova, R.R., Sigaeva, N.N., Kukovinets, O.S., Volodina, V.P., Spirikhin, L.V., Zaidullin, I.S., Kolesov, S.V., 2014. Modification of hyaluronic acid and chitosan, aimed at developing hydrogels for ophthalmology. Russ. J. Appl. Chem. 87, 1547–1557. Wang, X., Dai, W.-W., Dang, Y.-L., Hong, Y., Zhang, C., 2018. Five years’ outcomes of trabeculectomy with cross-linked sodium hyaluronate gel implantation for chinese glaucoma patients. Chin. Med. J. 131, 1562–1568. Yui, N., Okano, T., Sakurai, Y., 1992. Inflammation responsive degradation of crosslinked hyaluronic acid gels. J. Control. Release 22, 105–116. Zeng, W., Li, Q., Wan, T., Liu, C., Pan, W., Wu, Z., Zhang, G., Pan, J., Qin, M., Lin, Y., Wu, C., Xu, Y., 2016. Hyaluronic acid-coated niosomes facilitate tacrolimus ocular delivery: Mucoadhesion, precorneal retention, aqueous humor pharmacokinetics, and transcorneal permeability. Colloids Surf. B 141, 28–35. Zhang, L., Li, Y., Wang, C., Li, G., Zhao, Y., Yang, Y., 2014. Synthesis of methylprednisolone loaded ibuprofen modified inulin based nanoparticles and their application for drug delivery. Mater. Sci. Eng. C 42, 111–115.

284–293. Sahiner, N., Sagbas, S., Sahiner, M., Ayyala, R.S., 2017. Polyethyleneimine modified poly (Hyaluronic acid) particles with controllable antimicrobial and anticancer effects. Carbohydr. Polym. 159, 29–38. Sahiner, N., Silan, C., Sagbas, S., Ilgın, P., Butun, S., Erdugan, H., Ayyala, R.S., 2012. Porous and modified HA particles as potential drug delivery systems. Micropor. Mesopor. Mater. 155, 124–130. Salwowska, N.M., Bebenek, K.A., Zadło, D.A., Wcisło-Dziadecka, D.L., 2016. Physiochemical properties and application of hyaluronic acid: a systematic review. J. Cosmet. Dermatol. 15, 520–526. Siafakaa, P.I., Titopouloua, A., Koukarasb, E.N., Kostoglouc, M., Koutrisd, E., Karavasd, E., Bikiaris, D.N., 2015. Chitosan derivatives as effective nanocarriers for ocular release of timolol drug. Int. J. Pharm. 495, 249–264. Stone, J.L., Robin, A.L., Novack, G.D., Covert, D.W., Cagle, G.D., 2009. An objective evaluation of eye-drop instillation in glaucoma patients. Arch. Ophthalmol. 127, 732–736. Suner, S.S., Ari, B., Onder, F.C., Ozpolat, B., Ay, M., Sahiner, N., 2019a. Hyaluronic acid and hyaluronic acid: Sucrose nanogels for hydrophobic cancer drug delivery. Int. J. Biol. Macromol. 126, 1150–1157. Suner, S.S., Sahiner, M., Akcali, A., Sahiner, N., 2019b. Functionalization of halloysite

11