Alginate Hydrogels as Injectable Drug Delivery Vehicles for Optic Neuropathy Treatment

Optic neuropathy is the loss of visual acuity following damage to the optic nerve (ON). Traumatic optic neuropathy (TON) occurs when the optic nerve is injured following blunt or penetrating trauma to either the head or eye. Current management options for TON include the systemic delivery of corticosteroids and surgical decompression of the optic nerve; however, neither option alleviates the generation of reactive oxygen species (ROS) which are responsible for downstream damage to the ON. Addressing this limitation, an injectable alginate hydrogel system was developed to act as a drug delivery vehicle for methylene blue (MB), a confirmed ROS scavenger and neuroprotective agent. This MB-loaded polymeric scaffold has the ability to be injected as a liquid and rapidly form a gel around the optic nerve following the primary injury, allowing for the prolonged release of MB. The MB-loaded alginate hydrogels demonstrated minimal cytotoxicity to human retinal pigment epithelial (ARPE-19) cells and facilitated gradual MB release over 12 days. Additionally, the MB concentrations displayed a high degree of ROS scavenging after release from the alginate hydrogels, suggesting our approach may be successful in reducing ROS levels following ON injury. Graphical Abstract


Introduction
Optic neuropathy results in visual dysfunction due to optic nerve damage (e.g. glaucoma) and can be caused by several mechanisms. Traumatic optic neuropathy (TON) is an ocular injury in which the force or motion of the globe or orbital tissues is transferred from the eye or skull to the optic nerve (ON). It can be characterized by transient or permanent vision impairment, associated vascular and edema damage and subsequent ON atrophy [1]. In civilians, 0.4% of trauma patients incur TON; this increases to 5% in cases involving closed-head injuries [3]. Among soldiers, its incidence is higher, with ocular trauma representing up to 13% of recent battlefield injuries [4].
The current management options for the treatment of TON include the systemic delivery of corticosteroids and surgical decompression of the nerve. However, both treatments are ineffective at improving visual recovery, have side effects such as optic atrophy, complications following surgical decompression [5], and do not address secondary injury mechanisms such as the unattenuated generation of reactive oxygen species (ROS) [6]. Prolonged exposure to high ROS levels in the ON can cause a multitude of cellular dysregulations, namely the migration of inflammatory cells to the site of injury. These secondary injuries contribute to the formation of glial scarring [7], preventing tissue recovery by inhibiting signal transduction following the primary injury and contributing to permanent vision loss. Therefore, there is a demonstrated need to develop an effective treatment to sustain the release of ROS scavengers, thereby addressing the injuries caused by secondary injuries following TON.
Methylene blue (MB) is a potent ROS scavenger and neuroprotective agent capable of crossing the blood-brain barrier with a demonstrated inhibitory effect on glial cell migration in vivo [8 -12]. These qualities, along with recent discoveries of recovery following traumatic brain injury (TBI) and stroke in rats [13,14], makes MB a promising therapeutic candidate for TON treatment.
Due to its small molecular weight and high-water solubility, direct local injection of MB to the ON would be inadequate, leading to rapid diffusion of MB away from the site of injury. By surrounding the injured nerve with a MB-loaded degradable scaffold, drug elution would be better facilitated, allowing for a more sustained therapeutic release.
Alginate-based hydrogels were selected as the drug delivery vehicle in our study due to their biocompatible nature and extensive usage in tissue engineering and drug delivery applications [15,16]. Typically, aqueous alginate is externally crosslinked by the addition of dissolved Ca 2+ ions, forming a hydrated scaffold. This method was initially investigated for our TON treatment, but it induced rapid gelation, limiting its injection feasibility through a small gauge needle. Draget et al. [16], introduced an internal method of alginate crosslinking ( Figure I), in which insoluble calcium carbonate (CaCO3) particles are evenly distributed throughout the alginate solution before the addition of a slow hydrolyzing proton donor, in our case, D-glucono-lactone (GDL) [17]. The internal method of crosslinking slows the process of gelation to a rate that accommodates mixing of all components, subsequent injection through a small gauge needle and in situ hydrogel formation. By modulating the concentrations of the hydrogel's constituents, we developed a hydrated polymeric scaffold with tunable capabilities. Previous studies have evaluated alginate gels crosslinked with calcium carbonate as an injectable vehicle for osteoblast delivery in tissue engineering applications [19 -21]; however, an extensive evaluation of its drug delivery capabilities has yet to be studied. Given the need for a TON treatment that addresses the current limitations, internally crosslinked MB-loaded injectable alginate hydrogels could potentially lower the concentration of ROS and effectively improve visual recovery following injury.
With the above considerations, the purpose of this study was to synthesize alginate hydrogels loaded with MB through internal crosslinking and in situ gelation. We hypothesized that MB stimulated by oxidative stress could achieve ROS scavenging and effectively halt the generation of deleterious reactive species.  [22] (A) Alginate is a polysaccharide copolymer composed of two residues, (1-4)-linked β-D mannuronate (M), and α-L-guluronate (G). The patterning and ratio of these residues can significantly impact the material properties of hydrogels. (B) Schematic of the crosslinking reaction between the proton donor D-glucono-lactone (GDL), the calcium ion source CaCO3 and the alginate polymer. The reaction generates three products -gluconic acid, carbon dioxide, and the calcium ion-alginate complex. (C) [18] Once Ca 2+ is freed by GDL, the free ion interacts with alginate's carboxyl group to from ionic crosslinking between polymers.

Hydrogel Synthesis
Sodium alginate hydrogels were synthesized based on methods reported in literature with modifications [17]. 21 total hydrogel formulations were prepared and evaluated using design of experiments to modify alginate, CaCO3 and GDL concentrations. The first 9 formulations were selected based on their consistency in forming solid homogeneous hydrogels (Table I). They served as the models for pH testing in preliminary experiments (see Section 2.3).
Briefly, sodium alginate (0.63% -1.85% final w/v in gel) was dissolved in DI H2O by vortexing for 30 seconds and heating in 37°C water for 24 hours. Aqueous 1 mg/mL MB was added to a final concentration of 0.05 mg/mL, followed by the addition of CaCO3 and vortexed. As gelation is initiated rapidly following addition of GDL, the solution was quickly transferred to a mold or onto the rheometer stage following subsequent mixing of all components.
To prepare the remaining hydrogel formulations (10 -21), GDL:CaCO3 molar ratio concentrations were based off of the original 9 formulations that exhibited a neutral pH of 7.0 ± 1.0 (Figure II, Table I). These GDL:CaCO3 ratios ranged from 0.125 -1.00. The Ca 2+ :alginate monomer molar concentrations were also evaluated as a factor and varied from 0.5 -1.5. The ratios were selected to assess the influence of alginate and crosslinker concentrations on drug release, cytotoxicity and viscoelastic properties. Hydrogel formulations 10, 16 and 21 were selected as low, medium and high concentration hydrogels due to their GDL:CaCO3 molar ratios; 0.125, 0.500 and 1.00, respectively. They were analyzed further in cytotoxicity and ROS experimentation (see sections 2.4, 2.5). Table I. Composition of hydrogel formulations prepared and evaluated. The alginate hydrogels were designed based on their ability to form solid homogenous hydrogels. Formulations consist of 180 mg sodium alginate with varying molar concentrations of CaCO3 and GDL. The final GDL and CaCO3 concentrations were modulated based on preliminary hydrogels (1 -9). Molar concentrations of GDL:CaCO3 ratios ranged from 0.125 -1.00. Aqueous 1 mg/mL MB was also added to each formulation., except in ROS studies in which MB ranged from 0.05 -2.0 mg/mL to access the influence of MB concentration on ROS scavenging ability.

Hydrogel pH
The pH values of alginate hydrogel formulations 1 -9 were evaluated using a calibrated pH probe (Mettler Toledo, InLab Expert Pro-ISM, Columbus, OH) for 72 hours to evaluate pH evolution and determine the final compositions of hydrogel formulations 10 -21. The final pH was reported as the equilibrium pH (pHE).

Hydrogel Cytotoxicity
The biocompatibility of representative low, medium and high concentration hydrogels (formulations 10, 16, 21) were evaluated using the MTS assay and adapted from the methods of Niu et al. [24]. ARPE-19 cells were first seeded at 5 × 10 3 cells per well in a 96 well-plate and incubated for 24 hours in 200 µL base media (DMEM/F12, 10% FBS, 1% PS). 1 mL hydrogels were formed in 15 mL conical tubes and allowed to completely gel for 72 hours before 60 -minute UV light exposure, ensuring sterility [25]. The hydrogels were then immersed in 1 mL base media for 24 hours before media collection. The cells were incubated in 200 µL samples for 48 hours prior to performing the MTS assay. A positive control of base media, negative control of 1:9 dimethyl sulfoxide (DMSO): growth media [26] and blank of phenol-free DMEM were used to validate the assay. After incubation, the hydrogel-soaked media was removed and each well washed three times with 200 µL DPBS. Following, 180 µL of phenol-free growth media and 20 µL MTS reagent was added to each well and allowed to incubate for 1 hour. Optical density (OD) of the MTS-treated media was measured at 490 nm using a BioTek Elx808 plate reader (Winooski, VT). The cells were washed with DPBS once and the excitation and emission wavelengths; 485 nm and 535 nm, respectively, were measured using a microplate reader.

ROS Scavenging
To further confirm the ROS scavenging ability of MB, 1 mL hydrogels (formulations 10, 16,21) were formed in 15 mL conical tubes and allowed to gel for 72 hours. Following gelation, the hydrogels were exposed to UV light for one hour to sterilize. The hydrogels were then immersed in 1 mL base media for 24 hours before media collection. ARPE-19 cells were seeded on a 96 well-plate with 2 × 10 4 cells per well in base growth media and allowed to grow for 24 hours. The culture media was removed and the hydrogel -soaked medium was added to the wells. Hydrogen peroxide (10 µL, 600 µM final concentration) was added to test wells while DPBS was added to the other wells as a negative control. Additionally, hydrogel formulation 16 (medium concentration hydrogel) without MB was included as a negative control. Cells were incubated for 24 hours. Following incubation, the media was removed and 100 µL of DCFH-DA solution was added to each well and incubated for 1 -2 hours. The cells were thoroughly washed with PBS, and the excitation and emission wavelengths were measured at 485 nm and 535 nm, respectively.

Gelation Kinetics and Mechanical Properties
Oscillatory shear rheology was used to characterize the gelation kinetics, strain amplitude response, and frequency response of alginate hydrogel formulations 10 -21 [22]. The rheometer used was a Malvern Panalytical Kinexus Ultra+ (Malvern, United Kingdom) with a 20 mm titanium parallel plate upper geometry (PU20 SW1511 TI) and aluminum lower geometry (PLC61 S3722 AL). For all rheological tests, the gap height between the lower and upper geometries, the temperature and sample size were kept constant at 1 mm, 37°C and 375 µL, respectively.
To measure the gelation kinetics of alginate hydrogels, the alginate solution was dispensed as a liquid directly onto the lower geometry of the rheometer immediately following the addition and mixing of GDL. A constant frequency and strain amplitude of 1 Hz and 1% respectively (within linear viscoelastic region), were applied to the sample with its resulting shear stress measured every 5 seconds for 2 hours. The gelation time was defined as the time which gelation had terminated and was determined from the constant frequency and strain test as the first timepoint where complex shear modulus (G*) did not increase by more than 1% of the average of the 10 previously collected measurements [22]. A frequency sweep test immediately followed the gelation test, evaluating the frequency response of the hydrogel. Here, a constant strain amplitude of 1% was applied to the sample while frequency increased from 1 Hz to 100 Hz. The stiffness of the hydrogels is reported as the value of G* at 1 Hz from frequency sweep tests.
Representative low, medium, and high concentration CaCO3 and GDL hydrogels (formulations 10, 16, and 21, respectively) were additionally subjected to an amplitude sweep test to evaluate strain amplitude response. A constant frequency of 1 Hz was applied to the sample while the strain amplitude increased from 0.1% to 100%, and resulting stress was measured. Hydrogels were then immersed in 1 mL DPBS modified without calcium chloride (CaCl2) and magnesium chloride (MgCl2) at 37°C and at regular intervals (0, 1, 3, 7, and 14 days), DPBS was removed and the mass of the hydrogels was recorded. Results were calculated according to the following equation:

Hydrogel Swelling and MB Release
Here, Q is the swelling ratio, Ms is the mass of the formed hydrogel following incubation in DPBS at 37°C and excess water removal and MD is the mass of the 1 mL alginate solution placed in the tube [23].
The release kinetics of MB were evaluated using the same formulations (10 -21) evaluated for swelling. 1 mL hydrogels (10 -21) loaded with 1 mg/mL MB were created. Following immersion in DPBS and incubation at 37°C, 1 mL DPBS was removed at the given intervals (0, 1, 3, 7, and 14 days). 100 µL samples of the DPBS were placed in a 96 well-plate and absorbance measured.
The concentration of MB remaining in hydrogels following DPBS incubation was then determined using a standard concentration-absorbance curve measured at 630 nm using a plate reader (BioTekElx808).

Hydrogel Degradation
1 mL hydrogel solutions based on formulations 10 -21 were cast in pre-weighed 15 mL conical tubes and weighed. After incubation at 37°C for 72 hours, excess water was removed from tube and hydrogels were weighed again to determine weight following incubation. Hydrogels were immersed in 10 mL 1X DPBS with MgCl2 and CaCl2 at 37°C for 0, 1, 3, 7 or 14 days. At each timepoint, the DPBS was removed, the hydrogels were frozen at -80°C for 24 hours and lyophilized for 24 hours. Hydrogel degradation was reported as the percentage change in the mass of dry components used to create the hydrogel to the dried hydrogel mass after freezing and lyophilization.

Statistical Analysis
Data analysis was performed using two-tailed student t-test. Statistical significance was defined as p < 0.05. All values and data points are reported as the average ± standard deviation.

Hydrogel pH
The pH of hydrogel formulations 1 -9 was recorded for 72 hours ( Figure

Rheological Characterization
As shown in Table I, alginate hydrogels 10 -21 were prepared by varying CaCO3 and GDL concentrations. Time sweep rheology analysis ( Figure III, Table II) found that different concentrations of the hydrogel components had an observable influence on complex shear modulus (G*). As the concentrations of both CaCO3 and GDL increased, the complex shear modulus also increased. Increasing GDL content significantly increased G* more than the addition of CaCO3.
Additionally, complex modulus was dependent on GDL:CaCO3 ratios. Lower ratios corresponded lowered moduli and vice versa. All gelation times for formulations excluding 11 and 14 were significantly different from each other (p < 0.05). Gelling time was found to be tunable, decreasing with higher concentrations of both GDL and CaCO3. All hydrogels exhibited a storage modulus significantly greater than their loss moduli and had a G* of at least 35 Pa at 1 Hz.   Immediately following the gelation test, a frequency sweep was run on each hydrogel sample in triplicate with the result reported as the average ± standard deviation (n = 3). Figure   Formulation 21 demonstrates a sharp increase in complex shear strain from followed by a decrease around 20% complex shear strain, indicative of "fracturing."

Swelling and MB Release of Alginate Hydrogels
The swelling and MB release profile of the hydrogels was recorded in vitro over a period of 14 days ( Figure VIB and VIIA, respectively). The degree of equilibrium swelling varied among hydrogels, ranging from 0 -150%. Formulations 12 and 21 had the lowest and highest swelling percentage, respectively, correlating to low and high GDL:CaCO3 ratios. The degree of swelling varied is indicative of the components within the hydrogels. Low to medium concentration (of both CaCO3 and GDL) hydrogels had degrees of swelling reported around 100-120%, whereas high concentration hydrogels had swelling above 120%. MB release from the hydrogel formulations is further detailed in Figure VI. Among all hydrogels, an initial burst release was observed within the first 5 days, with over 50% MB released. Following the initial burst, a slower and more sustained release followed until the hydrogels disintegrated.
Lower concentration hydrogels had the most cumulative MB release (~90%) by 12 days, the point at which the alginate hydrogels were mostly dissolved and released remaining MB.

Cytotoxicity of Alginate Hydrogels
A fundamental requirement for injection is minimal cytotoxicity. To this end, following synthesis of representative hydrogels, we studied their biocompatibility using a human retinal cell line. MB Release (%) Formulation B results using the different crosslinker further support biocompatibility as well as their potential for injection. Figure VIII. Cytotoxicity of Representative Hydrogels. Cellular viability as measured by optical density (OD) of the MTS reagent product following exposure to alginate hydrogels. The low, medium, and high concentration hydrogels that were evaluated maintained a cell viability of at least 70% that of the positive control base media (DMEM/F12, 10% FBS, 1% PS).

MB as a ROS Scavenger
Scavenging of ROS by MB was evaluated through in vitro testing based on published methods [27]. ARPE-19 cells were first incubated with MB concentrations of 0, 0.05, 0.25, 0.50, 1.0 and 2.0 mg/mL for 24 hours and then treated with H2O2 for 24 hours. ROS levels/activity was characterized by the appearance of highly fluorescent compound DCF in the DCFH-DA assay.
There was an observable decrease in fluorescence of the cells corresponding to increased MB concentrations. We confirmed that ROS levels decreased significantly with concentrations of 0.500, 1.00 and 2.00 g/L (p < 0.05) ( Figure VIII). These results suggest the potential of using MB as ROS scavengers for TON treatment. The ability to scavenge ROS was confirmed with MB. Additional studies were performed with alginate to further confirm MB's ROS scavenging ability while loaded into a hydrogel. All hydrogels were loaded with 1.0 g/L MB, except the negative control 16, which as loaded without MB. ARPE-19 cells were incubated with hydrogel formulations 10, 16, 16 without MB and 21 for 24 hours. Following incubation, the hydrogels and cells were exposed to H2O2 for 24 hours with resulting DCF fluorescence measured. Hydrogels 10 and 21 displayed higher degrees of cell survival compared to 16, yielding similar results to our cytotoxicity study. Low and high concentration alginate hydrogels (10, 21) achieved ARPE-19 survival of over 60% when exposed to the highly cytotoxic H2O2. Medium concentration hydrogels (16) maintained cell survival of ~35% (with MB); however, survival was lowered to ~10% when cells were exposed to the hydrogels without MB. The presence of MB was found to significantly influence cell survival when loaded into hydrogel formulation 16 (p < 0.01) as survival increased from ~10% without MB to 35% with MB. Cell survival was maintained at over 50% for formulations 10 and 21. Cell survival did decrease following exposure to H2O2. Differences between 10 and 21 were found to not be statistically significant (p > 0.05). Differences between formulation 16 with and without MB was found to be statistically significant (p < 0.01).

Hydrogel Degradation
Naturally derived biomaterials can be advantageous for drug delivery applications as their components can be broken down and removed by the body. Biodegradation of alginate can be more challenging than other biomaterials as it degrades by ion exchange. The in vitro degradation of alginate hydrogels was studied for two weeks with the mass of the initial and final mass recorded. Table III

Discussion and Conclusion
In this study, to address the shortage of treatment options for TON, we developed an injectabledrug loaded delivery vehicle to release MB around the optic nerve following injury. Sustainable MB release was achieved among all synthesized alginate hydrogel formulations and may be able to be used for optic neuropathy treatments.
As reported in Table I [29] which is evident in our results. Future studies may focus on increasing Ca 2+ concentrations to higher concentration levels to increase modulus. Additionally, higher concentration hydrogels gelled significantly faster than low and medium hydrogels (p < 0.05), with formulations 10 and 21 having the slowest and most rapid gelation time, respectively. ARPE-19 cells were used to represent the microenvironment of the optic nerve because although primary ON cells would be preferred, their usage is limited due to difficulty in obtaining/isolating and slow proliferation rates [30]. As such, the immortalized cell line ARPE-19 was chosen in our study. Cytotoxicity results indicated that the hydrogels demonstrated low to minimal toxicity with low and high GDL:CaCO3 concentration hydrogels preferred over medium hydrogels. Medium concentration hydrogels were found to be more cytotoxic than low and high concentration hydrogels (70% survival compared to >90%) potentially due to the Ca 2+: alginate molar ratio crosslinking maximum. Results are consistent with previous reports of the use of alginate hydrogels and MB for drug delivery and tissue engineering applications [8 -12,15,16]. In conclusion, to improve upon the current management options for TON, internally crosslinked sodium alginate hydrogels were developed. We also identified the hydrogel with optimal mechanical properties and drug release by modulating its components using design of experiments.
The designed alginate drug delivery system is biocompatible and adequately lowers the concentration of reactive oxygen species in vitro. Most importantly, the proposed design improves upon the current treatments for TON. Given the results of the drug release as well as its biocompatibility and injectability, high concentration alginate hydrogels have the potential to improve TON damage as well as other diseases in which there is an accumulation of reactive species, which will be validated in vivo in future studies.