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21 February 2026

Development of Autologous Serum Ocular Insert for Chronic Dry Eye Disease

,
,
and
1
Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA 15213, USA
2
Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15261, USA
3
Department of Clinical and Translational Science, University of Pittsburgh, Pittsburgh, PA 15213, USA
4
Department of Chemical Engineering, University of Pittsburgh, Pittsburgh, PA 15261, USA
Pharmaceutics2026, 18(2), 267;https://doi.org/10.3390/pharmaceutics18020267
This article belongs to the Special Issue Ocular Drug Delivery Systems and Formulations
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Abstract

Background: Dry eye disease is a multifactorial disease of the ocular surface and/or tear film. It is one of the leading causes of ocular morbidity worldwide. Current therapy primarily consists of topical application of artificial tears and anti-inflammatory drugs. Autologous serum eye drops are an alternative treatment typically reserved for severe dry eyes mainly due to the limitations associated with access, storage, and the need for frequent application. Methods: Herein we describe the design and characterization of a bilayer carboxymethylcellulose/serum ocular insert that may expand the utility and accessibility of this treatment method. The insert, designed to be placed in the inferior fornix of the eye, has a unique carboxymethylcellulose backing layer to enhance comfort and direct protein release to the ocular surface. Results: Released serum proteins were able to protect corneal cells in vitro after treatment with hydrogen peroxide, demonstrated by a significantly higher cell viability compared to both serum eye drops and untreated cells. Our in vivo studies showed that the ocular inserts were able to deliver epitheliotrophic growth factors to treated animals at a level similar to standard serum eyedrops at an 8-fold reduction in dosing frequency that was well-tolerated in the treated eyes. In comparison to the control, serum ocular inserts demonstrated improvement in dry eye signs and symptoms in a rabbit model. Conclusions: Our results demonstrate that the novel inserts prolong the delivery of key proteins and growth factors for treating dry eye disease and significantly enhance shelf stability.

1. Introduction

Dry eye disease (DED) is a chronic condition of the preocular tear film that results in symptoms of visual disturbance, tear film instability, and discomfort that can be extreme in severe forms of DED. It is usually associated with corneal and conjunctival inflammation, increased tear film osmolarity and neurosensory abnormalities [1,2,3,4,5,6]. DED has a wide prevalence, affecting over 16 million adults in the US alone, and its prevalence is expected to increase as the population ages, which may be up to 75% in adults over 40 years old [2]. The cost of treating DED presents a huge burden on the healthcare system, with an estimate of nearly $4 billion in direct annual expenditure on prescription drugs, over-the-counter products, and punctual plug placement [7,8]. Current treatment approaches include conservative options like artificial tears, ocular lubricants, lid hygiene, and lifestyle and environmental modifications [4]. However, artificial tears or ocular lubricants may include preservatives or excipients that cause irritation and ultimately worsen DED symptoms [7].
Inflammation plays a major role in the etiology and progression and severity of DED [9,10,11]. Therefore, pharmacological therapies like corticosteroids, cyclosporine A [12,13] and lifitegrast [14,15] work mainly by reducing the underlying inflammation to address DED symptoms. When these therapeutic modalities fail to control the progression of disease, one appealing alternative is the use of blood products, most notably autologous serum eye drops (ASEDs), which have similar composition and biochemical properties to native tears [16,17]. Normal tear film is an aqueous phase containing high mucin concentration in the inner layer and a thin superficial lipid film [18]. DED is usually associated with dysfunction of the lacrimal gland, resulting in tear deficiency accompanied by reduced lipid layer production from the meibomian gland, which exacerbates tear evaporation [19,20]. In this context, serum contains lipids, electrolytes, vitamins, and is isosmotic in comparison to the aqueous part of the tear film, around 300 mosm/L [16]. Additionally, serum contains epitheliotrophic factors like epidermal growth factor (EGF), transforming growth factor-beta1 (TGF-β1), lactoferrin, hepatocyte growth factor (HGF), and platelet-derived growth factor-AB (PDGF-AB). These factors play an important role in the expression of goblet cells and mucin production, formation of important extracellular matrices, and chemotactic effects that promote corneal epithelial healing [21]. Therefore, autologous serum has been a recent therapeutic choice for a variety of chronic diseases such as chronic urticaria [22], osteoarthritis [23] and infectious disorders [24]. ASEDs have been repeatedly shown to achieve a beneficial effect in advanced and severe cases of DED [16,25,26]. ASEDs are generally recognized to be more efficient than artificial tears, with fewer side effects than corticosteroid or cyclosporine A eye drops [12].
Despite these advantages, ASEDs present significant challenges in preparation, storage, and administration. First, there is a lack of standard approach for preparing and storing ASEDs; variations exist from clinic to clinic, which presents a barrier to obtaining predictable and reproducible results [25,26,27]. Another major disadvantage of ASED therapy is the need for frequent drop instillation for ongoing treatment [28,29]. Patients must self-administer ASEDs every 2–3 h for optimal results due to the short duration of physiological action of serum proteins [30]. Storage is equally problematic; bottles must be stored frozen until the day of use, refrigerated during that day, and disposed of at the end of the day with any remaining serum [25]. Additional studies have suggested that lyophilization can dramatically increase the shelf life of prepared ASED, but this effect is negated once eye drops have been reconstituted for use, and it fails to address the frequent dosing needs [31,32]. One of the main challenges in the ocular delivery of proteins is the chemical and physical instability due to denaturation, adsorption, aggregation, and precipitation that may lead to the inactivation of polypeptides in tissues [33]. A series of novel strategies such as glycoengineering, PEGylation, Fc-fusion, chitosan nanoparticles, and liposomes improved the efficacy, safety, and stability of peptide delivery to the eye, which consequently expanded the therapeutic potential of proteins [34,35]. An approach to overcome the aforementioned issues with ASEDs could be the development of a solid, lyophilized dosage form able to protect the therapeutic proteins and maintain prolonged release on the ocular surface. Accordingly, in this study, we present the preparation of solid, bilayered matrices of lyophilized serum and carboxymethylcellulose (CMC) to be used as ocular inserts for treating severe DED.
We demonstrate herein that a single, autologous serum ocular insert (ASOI) can safely achieve the same level of protein delivery as a full daily course of ASEDs upon placement in the inferior fornix, as demonstrated in preclinical rabbit studies. We provide support for our hypothesis that this beneficial effect can be achieved alongside improved stability and simpler storage methods. This work represents a promising advancement for novel, effective, and convenient DED therapy with numerous enhancements over current options.

2. Materials and Methods

All materials were obtained from Millipore Sigma (St. Louis, MO, USA) unless otherwise noted. Animal testing protocols utilized for this research were approved by the University of Pittsburgh’s Institutional Animal Care and Use Committee (IACUC), which follows the guidelines set forth by the National Research Council’s Guide for the Care and Use of Laboratory Animals. The preclinical methods also align with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.

2.1. Preparation of Autologous Serum Ocular Insert (ASOI)

Lyophilized serum was obtained by freeze-drying commercially available, human male AB plasma for 5 days (Lot. H4522). Lyophilized serum (20 mg) and carboxymethylcellulose (CMC) (20 mg, Lot 0000257487) were then separately molded into a circular shape 6 mm in diameter using 1134 kg of weight for 10 min in a Carver two-post press. One each of the serum and CMC discs were then temporarily fused together using 2 µL of deionized water applied between the two surfaces, followed by once again compressing under 1134 kg of weight for 20 min to obtain a (1:1) CMC: serum insert. We refer to each of these bi-layered inserts as a single ASOI.

2.2. Characterization of ASOI

2.2.1. Swelling Index

ASOI swelling studies were performed in PBS (pH 7.4). Each insert was weighed and soaked in PBS for selected periods of time (5, 10, 20, 30, or 60 min). After immersion, the inserts were removed from PBS, excess surface water was removed using filter paper, and the inserts were weighed. The structural integrity of each insert was maintained for the duration of the test. The degree of swelling was calculated using the following equation: swelling index = [(Wt − Wo)/W0] [36,37], where Wt is the weight of the swollen insert after a predetermined period of time (t). Wo is the initial weight of the insert at time = 0. Swelling measurements were performed in triplicate at each time point.

2.2.2. Scanning Electron Microscopy

ASOI morphology was characterized using scanning electron microscopy (SEM), model JEOL JSM-6390LV (JEOL, Tokyo, Japan), operating at 15 kV. The samples were sputter coated in gold (PELCO SC-6) for 20 s and analyzed at a suitable acceleration voltage.

2.2.3. Differential Scanning Calorimetry

The thermal properties of the inserts were evaluated using differential scanning calorimetry (DSC). Measurements were carried out using a DSC 250 equipped with a RCS90 cooling system (TA instruments, New Castle, DE, USA). Samples (inserts, powder CMC, and powder serum) were packed in an aluminum crucible and heated at a rate of 25 °C/min. Nitrogen was used as a purge gas during the analysis at a rate of 20 mL/min. Samples were run in triplicate, and all specimens were heated from 25 to 250 °C.

2.2.4. In Vitro Release of Serum Proteins

Release of serum proteins from the ASOI matrices was investigated using Franz cells with an effective diffusional area of 0.636 cm2. A Durapore® PVDF membrane (25 mm diameter, 0.45 µm) was inserted between the donor and receiving compartments. The receptor medium was PBS (5 mL), stirred at 400 rpm at 32 °C. The donor phase consisted of an ASOI matrix soaked with 30 µL of MilliQ water to simulate the physiological conditions. The release system was sealed to avoid any evaporation from the release media. Samples (0.5 mL) were withdrawn at selected time points for analysis and replaced with fresh buffer. The quantity of released proteins was determined using a Pierce BCA protein assay kit (Pierce, Cambridge, NJ, USA) according to the manufacturer’s instructions. Samples were collected for 24 h and analyzed in triplicate.

2.3. Characterization of Protein Stability After Compression

2.3.1. Fourier Transformed Infrared Spectroscopysza

Fourier Transformed Infrared spectroscopy (FTIR) spectra of the insert and polymer powders (serum and CMC) were recorded using a Nicolet iS50 FTIR Spectrometer (Thermo Scientific, Madison, WI, USA). The instrument has a diamond crystal for single-bounce attenuated total reflectance sampling. FTIR spectra were analyzed over a wavelength range of 4000–650 cm−1 after the accumulation of 20 scans.

2.3.2. Dynamic Light Scattering

Dynamic light scattering (DLS) experiments were carried out using Zetasizer Nano S90 (Malvern Instruments, Malvern, UK) at 25 °C. Serum samples and dissolved inserts with concentration of 2 mg/mL in deionized water were analyzed at a scattering angle of 90°.

2.3.3. Size Exclusion Chromatography

Size exclusion chromatography (SEC) was performed using a Waters 600 with Column YMC pack diol 300, 5 µm, 300 × 6 mm (YMC America Inc., Devens, MA, USA). The mobile phase was 50 mM pH 7.4 phosphate buffer containing 400 mM sodium chloride in isocratic flow at a rate of 0.6 mL/min (LC-20AT, Shimadzu, Kyoto, Japan) and a column temperature of 25 °C (CTO-10AC VP, Shimadzu, Kyoto, Japan). Protein molecular weight standards were purchased from Millipore Sigma (Lot 69385) and reconstituted according to the manufacturer’s directions. A volume of 20 µL per each sample was injected, and potential aggregate and/or fragment formation in dissolved inserts was analyzed compared to the chromatogram of the freshly prepared serum solutions. The diode array detector wavelength was set to 280 nm (SPD-40 UV Detector, Shimadzu, Kyoto, Japan).

2.4. In Vitro Oxidative Stress Model

Human corneal epithelial cells (HCECs, Lot 1002043) were cultured in EpiGRO serum-free basal media supplemented to a final concentration of 5 μg/mL apo-transferrin, epinephrine, 1.0 μM extract P, 0.4%, hydrocortisone hemisuccinate, 100 ng/mL L-glutamine, 6 mM rh insulin, 5 μg/mL CE growth factor, and 1% penicillin-streptomycin solution and incubated at 37 °C in 5% CO2. The cells were passaged and cultured at approximately 80–90% confluency. HCECs from the third to tenth passages that exhibited normal morphology were used in the experiments.
To simulate the effects of oxidative stress, the HCECs were plated at a density of 5 × 103 cells per well in a 96-well plate. Cells were incubated with gradient concentrations (0, 0.002, 0.02, 0.2, 2, and 20 mM) of H2O2 for 24 h. We observed a 50% cytotoxicity rate for the 0.2 mM H2O2 incubation; therefore, this concentration was used to evaluate serum treatment in the oxidative stress cell model for the following cell experiment.
To compare the relative cell protective abilities of serum and bilayered ASOIs, HCECs (5 × 103 cells per well in 96-well plate) were pretreated with 100 µL of PBS, serum (1 mg/mL), presoaked serum inserts in media (1 mg/mL) at 37 °C for 24 h to allow cellular uptake of serum growth factors. The culture medium was then replaced with media containing 200 μM H2O2 and incubated at 37 °C for 24 h. The survival rate of HCECs was examined using the CCK-8 assay (Dojindo Laboratories, Kumamoto, Japan) [38,39].

2.5. In Vivo Studies

New Zealand white rabbits, six months old, were purchased from Envigo (Somerset, NJ, USA). The nictitating membrane of both eyes of each animal was resected to better mimic the human eye and to allow for long-term retention of ASOI in the lower fornix according to previously published methods [40,41]. Briefly, rabbits were anesthetized using 10 mg/kg ketamine and 1 mg/kg xylazine (Ketathesia; Henry Schein Animal Health, Dublin, OH, USA and AnaSed Injection; Lloyd Laboratories, Shenandoah, IA, USA, respectively). Following this, one topical eyedrop of 0.5% proparacaine (Bausch + Lomb, Bridgewater, NJ, USA) was instilled in both eyes. The nictitating membrane was held gently with forceps and surgically removed with a scalpel, then any remaining tissue was electrocauterized. Subsequently, one topical eyedrop of 0.3% tobramycin (Bausch + Lomb, Bridgewater, NJ, USA), and one topical eyedrop of 1% prednisolone acetate (Pacific Pharmaceuticals Inc., Rancho Cucamonga, CA, USA) was immediately instilled in both eyes. Tobramycin/prednisolone drops were administered once daily for 4 days to prevent infection and manage inflammation. Two kinds of rabbit models were used to compare ASOI performance and safety to standard serum eyedrops: a healthy model and a benzalkonium chloride (BAC)-induced dry eye model.

2.5.1. Preclinical Testing in Healthy Rabbit Model

In this model, one ASOI was placed in the right eye of each rabbit once daily by gently exposing the lower fornix and manually placing the insert. The paired eye received ASEDs every 90 min for a total of 8 drops per day as a positive control. ASEDs were prepared for each animal by drawing 8 mL of blood 4 days prior to beginning the study. Blood was collected into serum tubes, allowed to clot for 2 h, after which serum was isolated via centrifugation (1000 RCF, 10 min, 25 °C) and stored frozen until use. A total of n = 10 rabbits were used in this design, with an equal number of male and female rabbits. A summary of the study design and timeline for the healthy rabbit model is represented in Figure 1A.
Figure 1. Study designs and timelines for ASOI testing in (A) healthy rabbits and (B) BAC-induced DED model.
The following analyses were carried out on days 0, 1 and 5. Topical anesthetic (2 drops of 0.5% proparacaine) was administered at the start of each slit-lamp examination. Staining was accomplished with topically applied rose Bengal strips (Jorgensen Laboratories, Loveland, CO, USA). Ocular surface visualization was done using slit-lamp. Areas of rose Bengal staining indicate areas of keratoconjunctival damage, with varying severity being determined using a standard scoring [42]. Within the palpebral fissure, the exposed ocular surface was divided into three sections: nasal and temporal bulbar conjunctiva, and the central cornea. The intensity of staining in each section was scored by a masked observer up to three points per section, with a maximum possible score of nine points. Photos of the stained corneas were scored by an ophthalmologist blinded to their identity. Additionally, tear film samples were obtained using Schirmer strips placed in the inferior fornix for 3 min, immediately followed by soaking the strips in PBS (pH 7.4) for 2 h, at which time the strips were removed and samples were stored at −20 °C for further analysis. Tear film samples were analyzed using ELISA assay kits according to the manufacturer’s instructions for the following rabbit growth factor concentrations: lactoferrin (Lot. 241118, Biotang, Lexington, MA, USA), EGF (CAT#EKC38435, Biomatik, Kitchener, ON, Canada), TGFβ (Lot. L241015734, Biomatik, Kitchener, ON, Canada), IgA (Lot. V19136271, Biomatik, Kitchener, ON, Canada), HGF (Lot. L241015761, Biomatik, Kitchener, ON, Canada), and PDGF (Lot. 1107111424, Invitrogen, Thermo Fisher Scientific, Carlsbad, CA USA). Upon sacrifice, both eyes of each animal were enucleated, fixed with Davidson’s reagent and dehydrated in a graded ethanol series. Corneas were embedded in paraffin blocks, and sections of 5 µm thickness were obtained and stained with hematoxylin and eosin.

2.5.2. Preclinical Testing in Dry Eye Disease Model

The efficacy of the ASOI was tested in a benzalkonium chloride (BAC)-induced dry eye model, in which 4 total rabbits were tested. One eye was randomized to receive topical solution of BAC (0.1%) twice daily for 7 days to induce DED-like symptoms. The use of BAC leads to tear film instability due to elevated levels of proinflammatory cytokines in cornea and conjunctiva, epithelial cell apoptosis, and lowered mucin content [43]. On day 7, the same eye received a single ASOI. The other eye was used as a negative control. A summary of the study design and timeline for the BAC model is represented in Figure 1B.
Examinations were conducted on days 0, 3, 5, 7 and 8. Slit lamp examination was performed to evaluate the integrity of the epithelial surface of the cornea and conjunctiva. Animals were given topical anesthetic (2 drops of 0.5% proparacaine in both eyes) at the start of each slit lamp examination. Staining was achieved using topically applied fluorescein strips (Jorgensen Laboratories, Chennai, India), and the ocular surface was visualized under cobalt blue light. The intensity of staining in each eye was scored as previously discussed in healthy rabbit design [42]. Furthermore, after the rabbits were comfortably restrained, standard Schirmer test filter paper strips (Alcon Laboratories, Fort Worth, TX, USA) were folded at the 5 mm notch and inserted into the lower lateral one-third of conjunctival fornix and eyelids closed by gentle force for 5 min. Then, the length of the moistened paper was recorded. Upon sacrifice, both eyes in each animal were enucleated, fixed with Davidson’s reagent and dehydrated in a graded ethanol series. Corneas were embedded in paraffin blocks, and sections of 5 µm thickness were obtained and stained with hematoxylin and eosin.

2.6. Statistical Analysis

All data are reported as mean ± standard deviation (SD) for three measurements. In vitro and in vivo activity data were reported after statistical analysis based on one-way ANOVA followed by Tukey’s post hoc test. Statistical significance difference was noted when p ˂ 0.05.

3. Results

3.1. Characterization of ASOI and In Vitro Release Profile

ASOIs were produced as circular discs 6 mm in diameter, shown in Figure 2A. The results of the characterization studies are presented below. The swelling index of the insert is shown in Figure 2B. Inserts hydrated very quickly, reaching more than 50% of total hydration within 10 min. The effect of serum on swelling behavior is also presented in Figure 2B, which shows that water uptake was significantly greater in the samples containing serum compared to CMC alone.
Figure 2. (A) Representative images of ASOI. (B) Swelling index of CMC and ASOI in PBS. Values expressed as mean ± SD (n = 3). (C) Representative SEM image of autologous serum loaded insert showing serum side. (D) In vitro release kinetics of serum proteins from a single ASOI in PBS at sink-like conditions. Values expressed as mean ± SD.
A qualitative morphological characterization of the bilayered ASOIs was performed via SEM visualization, as shown in Figure 2C. The inserts showed a moderately smooth surface with no obvious crystallized or granular particles. A representative image of the lateral side demonstrates that the insert has smooth edges and an approximate thickness of 70 µm.
The amount of protein released from a single ASOI in PBS at sink-like conditions was determined in vitro. The cumulative kinetics of total protein released over 24 h from the compressed matrix are shown in Figure 2D. Protein release followed a biphasic pattern in which an initial burst release of 68.84 ± 1.65% can be observed in the first 8 h followed by a more gradual release of protein for the remaining 16 h. In total, ASOI released 88.49 ± 5.77% of the initial protein concentration (labeled as 100% in Figure 2D) in 24 h.

3.2. Effect of Compression on Serum Proteins

The preservation of protein native structure in the dried state can be directly correlated with the prevention of aggregation and protection of activity of labile proteins after rehydration and prediction of long-term stability with storage [44]. Figure 3A shows the FTIR spectra of serum both before and after compression. No obvious peak shift was observed between the spectra of serum with the respective compressed form, though there were minor differences between the intensities of certain peaks.
Figure 3. (A) Comparison of FTIR spectra of serum and compressed serum. Spectra cover the range of 550–3500 cm−1. (B) Aggregate levels in reconstituted 1—freeze dried serum 2—ASOI investigated using dynamic light scattering at 25 °C. Data are shown as intensity size distribution. (C) DSC thermogram of serum, CMC and ASOI heated from 25 to 250 °C. Heat flow downward for endothermic peaks.
Compression of proteins in the solid state reduces intermolecular distances and can potentially induce aggregations, both reversibly and irreversibly. The level of aggregates was examined in reconstituted samples using DLS and SEC. DLS analysis of size distribution of freeze-dried serum samples resulted in 3 peaks at 10.86, 73.74 and 435.0 nm, suggesting the presence of agglomerates in dissolved ASOI samples, shown in Figure 3B. ASOI dispersion shown in Figure 3B displays a shift of the peaks to a larger size at 655.8 and 4506 nm. Despite this shift, no noticeable agglomerates could be detected by SEC in the serum samples after compression. This suggests the existence of the serum albumin in its molecular form, with comparative average molecular weights shown in Table 1. Although this method did not allow for the detection of individual growth factor molecular weights, it can be reasonably assumed that non-albumin proteins had similarly low levels of aggregation.
Table 1. Molecular weight measurements for reconstituted freeze-dried serum and ASOI analyzed using SEC.
Figure 3C shows the result of DSC analysis on samples of serum, CMC, and the bilayered ASOI. CMC presented a broad endothermic peak at 46.19 °C due to evaporation of residual water. In addition, CMC samples showed a broad endothermic peak at 120.39 °C, which can be attributed to the glass transition of the polymer [45]. Serum showed a complex multimodal DSC profile (Figure 3C) in line with those previously reported for serum and plasma samples [46]. Peak analysis of the mean thermogram yielded a melting point temperature (TM) value of 54 °C that is not linked to individual protein components of the serum proteome [47]. Peaks that appear from 117 to 136 °C could be attributed to changes due to unfolding of the protein structure. The TM of the serum in the compressed ASOI formulation was found to be approximately 54.5 °C, demonstrating thermal stability relative to its pre-compressed state. Minor shifts in endothermic peaks of CMC and serum were observed in the ASOI data. It is worth mentioning that the presence of the serum and CMC in layered dosage form would diminish the probability of interaction and subsequent changes in thermal behavior [48].

3.3. In Vitro Activity on Corneal Epithelial Cells

In DED, the corneal epithelium is exposed to excessive oxidative stress [49]. We assessed the in vitro protective activity against reactive oxygen species (ROS) of ASOI in an H2O2-induced oxidative stress model using HCECs. Figure 4 shows the dose-dependent toxicity of H2O2 against HCECs with a half-maximal inhibitory concentration (IC50) value of 200 μM. To understand the cell protective effects of ASOIs, they were co-cultured with HCECs in media spiked with 200 μM of H2O2. The cell viability was evaluated with the cell counting kit-8 (CCK-8) assay to demonstrate HCEC survival under oxidative stress [39]. The ASOI showed superior cell protective effects against the cytotoxicity of H2O2, where they preserved the HCECs’ viability up to 89.71 ± 7.1% compared to serum at 73.01 ± 1.6%.
Figure 4. (A) Cytoxicity profile of H2O2 on HCECs. (B) Cell viability of HCECs tested by CCK-8 assay after indicated treatments under oxidative stress. *** p < 0.001.

3.4. In Vivo Testing of ASOI

3.4.1. Healthy Rabbit Study

We performed a comparative study of ASOI and ASED treatment in healthy rabbits to establish a baseline of biocompatibility and tolerability. Rose Bengal staining of the ocular surface, the results of which are shown in Figure 5A, is a standard diagnostic test in dry eye and other ocular surface disorders. Masked scores of staining intensities for all animals are shown in Figure 5B and demonstrate no statistical difference between eight times daily ASED and once daily ASOI treatment groups. In both groups, an average staining score below 2 was maintained for the full 5 days. This strongly suggests acceptable tolerability during ASOI administration and over the 24 h consecutive residence in the inferior fornix [50]. This result was confirmed qualitatively through regular animal observation, with all ASOIs being well-retained in the fornix and rabbits exhibiting no signs of distress or discomfort, as suggested by species-specific indicators including facial grimace [51]. Although facial grimace was not explicitly scored, orbital tightening (as indicated by partial or complete closure of the eyelid) was not observed at any time point.
Figure 5. (A) Rose Bengal staining images after treatment with ASED and ASOI for 5 days in healthy rabbit design, with the green arrow showing a representative area of staining. (B) analysis of staining score of (A). (C) Growth factors levels, IgA, lactoferrin, HGF, TGF, PDGF and EGF in tear fluid samples collected at 0, 1 and 5 days after treatment with ASED and ASOI. (D) H&E-stained corneal sections from sacrificed animals on day 5 in healthy rabbit model. Scale bar is 100 µm. Ordinary one-way ANOVA, ns (p > 0.05), * (p < 0.05).
Epitheliotrophic factors present in serum are known to play important roles in regulating cell proliferation, migration, and differentiation of the epithelial cells of the ocular surface [52]. In this study, we measured the concentrations of six factors of particular interest in dry eye disease in tear fluid samples of healthy rabbits treated with either ASED or ASOI: EGF, HGF, PDGF-AB, TGF-β1, IgA and lactoferrin [53]. Figure 5C shows the differences in concentrations between groups; in particular, the concentration of PDGF, TGF and lactoferrin at day zero, in addition to lactoferrin, which was significantly higher in the ASOI-treated group on day 5. All other factors were present at comparable levels to samples from ASED-treated rabbits at all time points. Of note, no HGF was detected in any of the female rabbits; therefore, these levels are reported only for the five male rabbits. Corneal sections from enucleated eyes at the end of the study (day 5) were evaluated for relevant physiological and structural changes, if any, as shown in Figure 5D. H&E staining of ASED-treated eyes showed normal physiological and anatomical structures, such as an intact surface epithelium on the conjunctival side of the section, with no signs of macrophage infiltration and inflammation [54]. Similar representative sections from ASOI-treated eyes show comparable anatomical features and lack of inflammation.

3.4.2. Dry Eye Disease Design

The therapeutic effects of ASOI in DED-like conditions were evaluated in a BAC-induced rabbit model of tear film insufficiency. We analyzed epithelial integrity via corneal staining with fluorescein, volume of tear secretions, and ocular physiology upon sacrifice via histological evaluation. One randomized eye was selected for treatment with 0.1% BAC twice daily for 7 days to establish DED [54] followed by ASOI administration on day 7, while the untreated eye was used as a control. Rabbit corneas were stained at 0, 3, 5, 7 and 8 days with fluorescein to monitor DED symptoms and efficiency of ASOI therapy. As the corneal staining images show in Figure 6A, corneas with DED-like symptoms in the BAC + ASOI group showed substantial increase in staining area over the first 5 days (p < 0.05). There was a marked reduction in staining intensity upon initiation of ASOI treatment on day 7 that was maintained on Day 8. The staining scores on Day 8 for ASOI-treated and negative control corneas were 0.5 ± 0.25, and 0, respectively, as shown in Figure 6B.
Figure 6. (A) Fluorescein staining images of control and after treatment with BAC and ASOI for 7 days in dry eye rabbit design. (B) Analysis of staining scores in (A). (C) Schirmer test results after 7 days of treatment. (D) H&E-stained corneal sections from sacrificed animals on day 8 in dry eye rabbit model. Scale bar is 100 µm. Ordinary one-way ANOVA, ns (p > 0.05), * (p < 0.05).
Tear fluid collection using Schirmer strips was done to detect the volume of tear secretions, as shown in Figure 6C. As expected, tear secretions were elevated on day 3 as a compensatory reaction to the inflammation triggered by BAC administration compared to baseline levels [55]. Importantly, tear volumes of DED-like rabbits were significantly increased after being treated by ASOI (Figure 6C), which positively correlated with the results from masked corneal stain scores.
Tissue samples were collected from sacrificed animals on day 8 for histological evaluation (Figure 6D). BAC + ASOI treated eyes sectioned and stained with H&E showed mild degeneration of the epithelium but no signs of macrophage infiltration or inflammation. ASOI-treated eyes showed anatomical features similar to untreated eyes.

4. Discussion

DED is a dynamic and complex disease that is often characterized by loss of ocular surface and tear film homeostasis [4]. To address the limitations of ASED stability and the need for frequent dosing, our group developed a bilayered formulation consisting of individual layers of lyophilized serum and solid CMC compressed into an ocular insert. The goal was to achieve comparable serum protein release over 24 h to a full day’s course of ASEDs with this ASOI. This work builds on extensive research into non-invasive sustained drug delivery systems aimed at enhancing patient and clinician experiences, as well as improving outcomes across a range of ocular disorders [28].
The bilayered structure of ASOI is one of its unique and useful features, with the active layer consisting of lyophilized serum and the comfort layer of CMC, which acts as a diffusion barrier to encourage protein movement to the ocular surface. CMC is a common polymer additive to enhance the comfort and lubricating properties of eye drops, and it was selected due to its biodegradable, biocompatible, and mucoadhesive properties [56]. Swelling capacity is a particularly useful metric in evaluating polymeric formulations like ASOI. Upon placement of the insert in the eye, the tear fluid diffuses through polymeric matrix, resulting in its swelling, which in turn induces polymer chain relaxation, drug dissolution, and subsequently, drug diffusion. Furthermore, the polymers must swell to start the formation of weak bonds that provide their bioadhesive properties [57]. The ASOIs hydrated quickly, likely because serum (a hydrophilic active ingredient) increases the swelling index of CMC. The multilayered nature of the inserts diminished the probability of hydrogen bond formation between the polymer and serum, which would negatively affect the swelling potential of CMC, which is commonly observed in drug dispersed matrices.
The application of compression force increases the inter-particulate hydrophobic interactions in addition to decreasing the void spaces between them [58]. This could explain the prolonged dissolution time of the insert over 24 h. Additionally, hydration of CMC results in swelling and increasing the viscosity of the donor chamber, which would slow the serum diffusion to the receiver compartment [59]. From these preliminary data, it is evident that the insert was able to release serum over 24 h, which could be used for once daily dosing. Nevertheless, the matrix has shown the ability to release the peptide in simulated physiological media. On the other hand, the ASOIs showed considerable potential for therapeutic efficacy in vivo. Inserts were prepared as 6 mm diameter circular discs with a thickness of approximately 70 µm. Ocular inserts investigated previously in preclinical and/or clinical trials demonstrated a thickness of 70–500 µm, suggesting that the ASOIs developed in this study would be easily retained in the inferior fornix of the eye [60].
We used FTIR spectroscopy to analyze the protein structure in the ASOI. In this context, the amide I region (1650 cm−1), which directly relates to the conformation of the polypeptide backbone, arises from the C=O stretching vibration. Furthermore, the amide region II (1550 cm−1) can also provide useful information about changes in the secondary structure derived from in-plane N–H bending. It is related to the type and number of structural elements, such as α-helix, β-sheets, and γ-turns. In the amide III band, at around 1200–1300 cm−1, coupled C-N stretching and N-H bending occur. The secondary structure can usually be deduced from analyzing these regions [61,62]. Our results show significant overlap in the regions of interest for the compressed serum compared to native serum, suggesting that the secondary structure is intact after fabricating the ASOI.
Aggregation is a significant concern for biotherapeutics, as it could trigger immunogenicity and a loss of biological activity [63]. DLS intensity signals directly increase with the size of a particle; the intensity-averaged size results in a value weighted toward the larger end of a size distribution. This parameter provides sensitivity for the detection of even a minute level of aggregates [44]. The DLS data reported herein suggest that compression may have increased the formation of aggregates. However, SEC results confirmed the preservation of the protein hydrodynamic diameter after compression. Therefore, the aggregates in reconstituted samples could be of a reversible nature, which disassemble over time or with shear [64].
The final characterization study was performed to determine thermal and physical changes in ASOI using DSC. The serum melting point was maintained, suggesting resistance to compression and additive-induced changes. There were shifts noted in the polymer glass transition and protein unfolding. This could primarily be explained by the presence of the physical mixture [65] of CMC and serum in DSC samples. Additionally, the compression force used to prepare the insert may increase the cohesive forces and reduce void spaces between the molecules [66], which may result in a change in the thermal behavior of the serum and protein. However, the layered insert would limit the interaction of serum and CMC and most likely result in greater structural stability of serum. From these data, we anticipate that a sufficient level of stability could be achieved by formulating serum in a bilayered ASOI. Our assessment evaluated the potential deleterious effects of compression and formulation with CMC on the physical and structural characteristics of released serum. This was followed by studying the activity of ASOIs in cell and animal models to evaluate their therapeutic potential.
Cell viability measures of ASOI in a DED-like model suggest substantial efficacy in protecting the cells from oxidative stress. This could be related to the release of growth factors that promote corneal cell healing [21], as well as a potential role of the CMC layer, which has been shown to augment antioxidant activity [56]. The strength of the ASOI approach was highlighted using two separate preclinical models. First, a healthy rabbit model was used to test the hypothesis that a single ASOI can deliver serum proteins at comparable levels to ASEDs at the prescribed dosing frequency of 8 drops per day. This study also served as our initial opportunity to demonstrate retention of the insert in the fornix with little to no ocular irritation. ASED served as a reference for ease of administration and tolerability, as determined by staining and observation of animal behavior. The lack of irritation is likely due to the use of biocompatible materials in ASOI fabrication. The use of a rabbit’s own serum diminishes the probability of immunogenic responses that are usually encountered with protein therapies [67,68]. Furthermore, CMC is a widely used polymer due to its biocompatible and mucoadhesive nature to enhance the viscosity of artificial tears and residence time of topical ocular formulations [69,70,71]. The size and shape of the insert also likely contribute to overall comfort and lack of foreign body sensation.
In the healthy rabbit study, differences in growth factor concentrations between bilateral eyes could be observed on day zero. This could be related to the reflex tears from the stimulated eye, which could result in lower concentrations of growth factors due to high fluid turnover in comparison to the contralateral eyes [72]. There were no significant differences at any time point between the two treatment groups (p > 0.05), ASED and ASOI, except for lactoferrin on Day 5 (p < 0.05). The results of this study showed that lactoferrin levels in the ASOI-treated rabbits were significantly higher than in ASED-treated rabbits. While many factors contribute to levels of epitheliotrophic factors, like age, sex, and underlying health conditions [73,74,75], this finding nonetheless suggests superior growth factor delivery from the single ASOI at an 8-fold reduction in dosing frequency. Overall, the ASOI was well-tolerated and retained, and it was able to alleviate the need for multiple dosing (as with traditional ASED) while delivering the same or higher levels of serum proteins.
The second preclinical study utilized a BAC-induced model of tear film insufficiency. ASOIs showed outstanding remedial function in reversing BAC-mediated ocular damage via inflammation, apoptosis, and oxidative stress [76]. Tear volumes of DED-like rabbits were significantly increased after being treated by ASOI (p < 0.05), which correlated positively with the results acquired from corneal staining scores. Previous studies have demonstrated a dose-dependent effect of autologous serum on the expression of mucins. Vitamin A is a component of autologous serum that is highly linked to the improvement in the degree of squamous metaplasia by stimulating the production of mucins, restoring the normal composition of tear film [77]. Goblet cells are also sensitive to the action of vitamin A, which increases both the number and size of these cells. Moreover, epitheliotrophic growth factors have a major role in the improvement of squamous metaplasia in patients treated with ASED [78]. Future studies will seek to expand upon the tear volume findings to determine specific effects on mucin production and tear film integrity, and potentially to develop methods to enhance the delivery of key factors like vitamin A.

5. Conclusions

A novel autologous serum ocular insert was successfully synthesized using polymer additives and a straightforward compression method. This insert is intended to serve as an alternative to aqueous serum eyedrops, which have been criticized for their frequent, inconvenient dosing requirements and poor stability. The design of the ASOI was informed by the aforementioned challenges to enhance shelf life and improve protein bioavailability on the ocular surface. Our hypothesis that such a treatment modality would provide comparable levels of serum proteins with significantly decreased dosing frequency has been confirmed. The results of these studies, including the inhibition of corneal inflammation and increased tear volume in a DED model, suggest that once daily administration of ASOI could effectively treat DED. Our in vitro results further suggest that the mechanism of action is related to protection of the cells from oxidative stress through intrinsic growth factors. The extensive characterization of the ASOI confirms that the serum proteins maintain their physicochemical properties and remain highly bioactive after compression. Future studies will seek to further elucidate the primary mechanisms by which the ASOI is acting as well as expand upon the translation potential with additional preclinical studies.

Author Contributions

H.A.: Conceptualization, methodology, validation, formal analysis, investigation, data curation, visualization, writing—original draft. A.C.: Methodology, validation, investigation. V.J.: Conceptualization, writing—editing and review, supervision, funding acquisition. M.V.D.: Conceptualization, methodology, writing—editing and review, supervision, funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This project was supported by The Hillman Foundation, The Eye and Ear Foundation of Pittsburgh, an unrestricted grant from Research to Prevent Blindness, NEI NIH CORE grant P30 EY08098, and the University of Pittsburgh’s Clinical and Translational Science Institute.

Institutional Review Board Statement

The animal study protocol was approved by the University of Pittsburgh’s Institutional Animal Care and Use Committee (protocol number: 24023039; date of approval: 15 February 2024).

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare no conflict of interest.

List of Abbreviations

AbbreviationDefinition
DEDDry eye disease
ASEDAutologous serum eye drops
ASOIAutologous serum ocular insert
EGFEpidermal growth factor
TGF-β1Transforming growth factor-beta1
HGFHepatocyte growth factor
PDGF-ABPlatelet-derived growth factor-AB
CMCCarboxymethylcellulose
HCECsHuman corneal epithelial cells
BACBenzalkonium chloride
ROSReactive oxygen species
FTIRFourier transform infrared spectroscopy
DLSDynamic light scattering
SECSize exclusion chromatography
DSCDifferential scanning calorimetry

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