Controlled release of corticosteroid with biodegradable nanoparticles for treating experimental autoimmune uveitis

Abstract

Noninfectious uveitis is a potentially blinding ocular condition that often requires treatment with corticosteroids to prevent inflammation-related ocular complications. Severe forms of uveitis such as panuveitis that affects the whole eye often require a combination of topical and either regional or systemic corticosteroid. Regional corticosteroids are currently delivered inside the eye by intravitreal injection (e.g. Ozurdex®, an intravitreal dexamethasone implant). Intravitreal injection is associated with rare but potentially serious side effects, including endophthalmitis, retinal and vitreous hemorrhage, and retinal detachment. Subconjunctival (SCT) injection is a less invasive option that is a common route used for post-surgical drug administration and treatment of infection and severe inflammation. However, it is the water soluble form of dexamethasone, dexamethasone sodium phosphate (DSP), that has been demonstrated to achieve high intraocular penetration with subconjunctival injection. It is difficult to load highly water soluble drugs, such as DSP, and achieve sustained drug release using conventional encapsulation methods. We found that use of carboxyl-terminated poly(lactic-co-glycolic acid) (PLGA) allowed encapsulation of DSP into biodegradable nanoparticles (NP) with relatively high drug content (6% w/w) if divalent zinc ions were used as an ionic “bridge” between the PLGA and DSP. DSP-Zn-NP had an average diameter of 210 nm, narrow particle size distribution (polydispersity index ~0.1), and near neutral surface charge (−9 mV). DSP-Zn-NP administered by SCT injection provided detectable DSP levels in both the anterior chamber and vitreous chamber of the eye for at least 3 weeks. In a rat model of experimental autoimmune uveitis (EAU), inflammation was significantly reduced in both the front and back of the eye in animals that received a single SCT injection of DSP-Zn-NP as compared to animals that received either aqueous DSP solution or phosphate buffered saline (PBS). DSP-Zn-NP efficacy was evidenced by a reduced clinical disease score, decreased expression of various inflammatory cytokines, and preserved retinal structure and function. Furthermore, SCT DSP-Zn-NP significantly reduced microglia cell density in the retina, a hallmark of EAU in rats. DSP-Zn-NP hold promise as a new strategy to treat noninfectious uveitis and potentially other ocular inflammatory disorders.

1. Introduction

Uveitis is a sight-threatening intraocular inflammatory disease responsible for 10–20% of legal blindness in Western countries [1, 2]. Noninfectious uveitis is more common than infectious uveitis in developed countries, accounting for approximately 79–87% of cases [3]. Uveitis can be classified as anterior uveitis, intermediate uveitis, posterior uveitis, or panuveitis depending on the primary site of inflammation [4]. Panuveitis involves inflammation in both front and back segments of the eye [4], which is difficult to treat. Corticosteroids are the frontline treatment for noninfectious uveitis [5–7], acting to inhibit inflammation primarily through interaction with glucocorticoid receptors to suppress the activity of NF-κB and AP-1 [8, 9].

Eye drops are the most common method for ocular drug administration, but less than 5% of the drug instilled through eye drops is typically absorbed into ocular tissue [10], and the portion able to reach the posterior segment of eye is typically minimal in large mammals, especially humans. Thus, corticosteroids administered through eye drops showed limited effects in treating noninfectious uveitis affecting the posterior segment (intermediate, posterior, and panuveitis) in humans. Systemic administration of corticosteroids requires high doses (oral prednisone 1 mg/kg/day) in order to achieve therapeutic drug levels in the eye, which may be accompanied by significant systemic side effects [11].

Intravitreal administration of corticosteroids allows drug to reach the posterior segment of the eye since the vitreous chamber is located between the lens and retina. Ozurdex® is a biodegradable ocular implant that continuously releases a poorly water-soluble corticosteroid, dexamethasone [12], that is used to treat noninfectious intermediate and/or posterior uveitis [9, 13, 14]. Intravitreal injection of Ozurdex® requires a large, 22 gauge, needle and is associated with rare but potentially serious risks related to intravitreal injection. We and others have shown that periocular, subconjunctival (SCT) injection of a water-soluble analog of dexamethasone, namely dexamethasone sodium phosphate (DSP), can provide high drug levels in both the anterior and posterior segments of the eyes of humans [15, 16] and animals [17]. However, water-soluble drugs like DSP are typically cleared from the SCT injection site within a few hours [15, 17], leading to short-lived benefits. Therefore, we hypothesized that development of a method to achieve continuous delivery of water-soluble DSP following SCT injection may be effective in treating all types of noninfectious uveitis.

Here, we prepared DSP-loaded nanoparticles (DSP-Zn-NP) with high DSP loading that was dependent on divalent cation zinc ion bridging of the negative charges on DSP and carboxyl-terminated PLGA. DSP-Zn-NP were characterized, including DSP release rate in vitro, and then tested for potential efficacy following SCT administration in a rat model of noninfectious uveitis, namely the rat experimental autoimmune uveitis (EAU) model.

2. Materials and Methods

2.1. Materials

Poly(D,L-lactic-co-glycolic acid; LA:GA ratio 50:50, acid terminated) (PLGA) with M_W of 5.6 kDa and 34 kDa, and PLGA (LA:GA 50:50, ester terminated) with M_W of 11 kDa were purchased from Lakeshore Biomaterials (Evonik, Birmingham, AL). Dexamethasone sodium phosphate salt (DSP) was purchased from MP Biomedicals (Santa Ana, CA). [³H]-labeled DSP (1 mCi/mL) was purchased from American Radio labeled Chemicals (St Louis, MO). Maxidex 0.1% dexamethasone ophthalmic suspension (preservative: benzalkonium chloride 0.01%; vehicle: hypromellose 2910 0.5%) and 0.1% DSP ophthalmic solution (preservatives: sodium bisulfite 0.1%, phenylethyl alcohol 0.25% and benzalkonium chloride 0.02%) were purchased from Alcon Inc. and Bausch & Lomb Inc., respectively. Interphotoreceptor retinoid binding protein (IRBP) peptide, R16 (residues 1177–1191, sequence ADGSSWEGVGVVPDV) [18], was purchased from Anaspec Inc. (Fremont, CA). Pertussis toxin from Bordetella Pertussis, complete Freund’s adjuvant, Pluronic F127, triethanolamine, ethylenediaminetetraacetic acid solution (EDTA), zinc acetate dihydrate and organic solvents were purchased from Sigma-Aldrich (St. Louis, MO). Ionized calcium binding adaptor molecule-1 (Iba1) antibody was purchased from Wako Chemical USA, Inc. (Richmond, VA).

2.2. Ex vivo trans-scleral penetration of DSP and dexamethasone

Fresh adult rabbit eyes were obtained from Pel-Freez Biologicals, LLC (Rogers, AR), and shipped on ice. The ex vivo trans-scleral transport assay was carried out using a Franz diffusion cell (PermeGear, Inc., Hellertown, PA) using freshly excised rabbit sclera with the episcleral side facing the donor chamber [19]. The Franz diffusion cells have a diffusional area of 0.20 cm² and a receiver chamber volume of 5 mL. 0.3 mL of dexamethasone 0.1% ophthalmic suspension (Maxidex®, Alcon) or DSP 0.1% ophthalmic solution (Baush & Lomb) were added to the donor chamber. PBS (pH 7.0) was placed in the receiver chamber, maintained at 37°C and stirred at 600 rpm. 0.4 mL of sample was taken from the receiver chamber at various time points and replaced with fresh PBS. The dissected rabbit sclera was observed with a Zeiss light microscope to ensure there were not observable macroscopic changes to the tissue integrity and continuity before and after the 6-hour diffusion experiment. Drug levels were measured by HPLC on a Shimadzu Prominence LC system (Kyoto, Japan) equipped with a Pursuit 5 C18 column (Varian Inc., Lake Forest, CA). The mobile phase consisted of acetonitrile/water (35/65 v/v) containing 0.1% trifluoroacetic acid (flow rate = 1 mL/min), and column effluent was monitored by UV detection at 241 nm for DSP. For measuring dexamethasone, the mobile phase consisted of acetonitrile/water (50/50 v/v) containing 0.1% trifluoroacetic acid (flow rate = 1 mL/min), and UV detection at 235 nm.

We then calculated the scleral permeability coefficient (P, cm/sec), which is a measure of the drug permeability that is independent of time and surface area. P was estimated using the following equation [19]:

$$P=\frac{1}{Cd\times SA}\times \frac{\mathrm{\Delta }Q}{\mathrm{\Delta }t}$$

SA is the exposed surface area for transport within the Franz cell (0.2 cm²), Q is the cumulative amount of the drug transported across the sclera at any time t under sink conditions with intensive stirring. Cd is the original drug concentration in the donor chamber. We assumed that we reached a steady state and Cd was approximately constant during our 6 hours experiment. Data represent the mean of 3 diffusion cells.

2.3. Preparation and characterization of DSP-Zn-NP

DSP was encapsulated into PLGA nanoparticles using a solvent diffusion nanoprecipitation method, as previously reported [17]. Zinc was added to increase DSP encapsulation and extend drug release time, and Pluronic F127 was added to stabilize the nanoparticle formulation and to potentially reduce inflammation following injection [20]. Briefly, DSP-zinc complexes containing 20 mg DSP were co-dissolved with 100 mg carboxyl-terminated PLGA in 2.5 mL THF, and the pH was adjusted to 7–8 using triethanolamine. DSP-Zn complexes have essentially no solubility in water, and are easily dissolved in THF (solubility >10 mg/mL). The solution was slowly injected using a Hamilton syringe into 50 mL of a 5% F127 aqueous solution under magnetic stirring (700 rpm). After complete removal of THF by rotoevaporation (Rotavapor RII, BÜCHI, Switzerland), 1 mL 0.5M EDTA dissolved in water was added to dissolve any unencapsulated DSP-zinc complexes, and the DSP-Zn-NP were collected by centrifugation at 8,000 g for 25 min. DSP-Zn-NP were washed with 5% F127 aqueous solution to maintain the F127 coating, and suspended in 0.4 mL of ultrapure water. In order to prepare non-coated DSP-Zn-NP, all the procedures are the same except that water was used in place of 5% F127 solution. DSP-Zn-NP size (number mean) and surface charge (as measured by ζ-potential) were determined using a Zetasizer Nano ZS90 (Malvern Instruments, Southborough, MA) after being diluted in 10 mM NaCl aqueous solution (pH 7.4). The morphology of DSP-Zn-NP was characterized by transmission electron microscopy (TEM) using a Hitachi H7600 microscope (Hitachi Co. Ltd., Tokyo, Japan).

Drug loading (DL) was determined by measuring the amount of DSP in a known weight of lyophilized DSP-Zn-NP. To measure the DSP content in DSP-Zn-NP, approximately 50 μL of nanoparticles were lyophilized, weighed and dissolved in 0.5 mL of acetonitrile. Subsequently, 1 mL of 50 mM EDTA was added to solubilize zinc-DSP complexes, and the DSP concentration in the solution was measured by reverse phase HPLC. Isocratic separation was performed on a Shimadzu Prominence LC system (Kyoto, Japan) equipped with a Pursuit 5 C18 column (Varian Inc, Lake Forest, CA) and mobile phase consisting of acetonitrile/water (35/65 v/v) containing 0.1% trifluoroacetic acid (flow rate = 1 mL/min). Column effluent was monitored by UV detection at 241 nm. The drug encapsulation efficiency (EE%) was the ratio of actual drug loading in comparison to theoretical drug loading. Drug release studies were carried out in vitro at infinite sink conditions. In brief, 400 μL of nanoparticle suspension was sealed in a dialysis tubing cellulose membrane (MW cutoff 10 kDa, Sigma Aldrich, St. Louis, MO). The sealed dialysis membrane was placed into a 50 mL conical tube containing 12 mL of release media (PBS, pH 7.4) and incubated at 37°C on a platform shaker (140 rpm). The entire release media was collected at predetermined intervals and replaced with another 12 mL of fresh PBS. Release experiments were conducted in triplicate. The DSP levels were measured by HPLC as described above.

2.4. Ocular pharmacokinetics

Drug levels in ocular tissues were quantified by measuring the radioactivity of [³H]-labeled DSP using a scintillation counter. [³H]-labeled DSP was blended with non-labeled DSP (5 μCi:1 mg DSP) for the preparation of DSP-Zn-NP. DSP-Zn-NP were then suspended in saline at a [³H] concentration of ~16 μCi/mL. DSP solution at the same concentration and blending ratio was prepared. Thirty μL (~0.5 μCi or 100 μg DSP per eye) of either DSP-Zn-NP suspension or DSP solution was administered to both eyes of the same Lewis rat (5 weeks old) through SCT injection. At predetermined time intervals after injection (2 h, 1 day, 3 days, 5 days, 7 days, 14 days and 21 days), blood from the tail vein, the aqueous humor (fluid in the anterior chamber between cornea and lens), the vitreous humor (fluid in the vitreous chamber between retina and lens), and the subconjunctiva tissue around the injection site were carefully collected (4 eyes at each time point). Samples were weighed and processed for radioactivity counting using a scintillation counter (Perkin Elmer, Waltham, MA). The level of DSP in the blood was calculated by averaging two rats per time point. The total percentage of the injected dose at the injection sites and the radioactivity per mg of tissue were calculated.

To investigate the effect of SCT injection on the fellow eye, we injected 30 μL (~0.5 μCi or 100 μg DSP per eye) of DSP-Zn-NP into one eye of Lewis rats (n=3). After one day, the blood and ocular tissues were collected and processed as described above.

2.5. Induction of EAU in Lewis rats and treatment

All experimental protocols were approved by the Johns Hopkins University Animal Care and use Committee. Female Lewis rats (5–6 weeks old; Charles River Laboratories), were housed under pathogen-free conditions. The animals were anesthetized by intramuscular injection of a mixture of ketamine (50 mg/kg) and xylazine (5 mg/kg) during all experimental procedures.

Rats were immunized through a subcutaneous injection of 200 μL of IRBP peptide R16 (500 μg/ml) emulsified in complete Freund’s adjuvant (1:1 v/v) distributed among the two footpads and the tail base. Concurrently, rats received an intraperitoneal injection of 0.3 μg pertussis toxin [21].

To investigate therapeutic effects, both eyes were treated at post-immunization day (PID) 8 with a single SCT injection of (1) 30 μL of DSP-Zn-NP (6 mg DSP/mL), n=12 rats, (2) 30 μL of DSP free drug solution (6 mg DSP/mL), n=12 rats, and (3) 30 μL of PBS, n=10 rats. Four rats each group were sacrificed at PID 12. Erythromycin ophthalmic ointment was applied to the cornea following SCT injection to prevent cornea drying.

2.6. Clinical observation

Eyes were examined daily for clinical signs of ocular inflammation using a binocular microscope (Carl Zeiss, Germany) with coaxial illumination until the end time point (PID 18). Dilated blood vessels, abnormal pupil contraction, opacity of the anterior chamber and the absence of red reflex were scored in a masked fashion on a scale of 0 to 4 (Table S1), according to established criteria [22]. The recorded clinical scores were listed in Table S2A,B,C.

2.7. Slit lamp and fundus imaging

Rats were anesthetized and their pupils were dilated with 0.5% tropicamide (Baush & Lomb Ltd, Tampa, FL) and 0.5% phenylephrine hydrochloride (Baush & Lomb Ltd, Tampa, FL). The anterior segment and the fundus were recorded with a SL120 slit lamp microscope (Carl Zeiss AG, Oberkochen, Germany) and a Micron II small animal retinal imaging AD camera (Phoenix Research Lab, Inc., Pleasanton, CA), respectively.

2.8. Measurement of IOP

Non-invasive intraocular pressure (IOP) measurement will be conducted weekly after SCT injection of DSP-NP using a rebound Tonolab tonometer (Icare, Helsinki, Finland) specifically designed for IOP measurement in rats. IOP recorded for each eye will be the average of three measurements.

2.9. Electroretinography (ERG)

Retinal function was evaluated by recording scotopic ERGs using a UTAS visual electrodiagnostic system and BigShot™ Ganzfield stimulator (LKC Technologies, Gaithersburg, MD). At PID 18, rats were adapted to the dark overnight, and all procedures were performed under dim red light. After receiving anesthesia, rats were kept on a heating pad to maintain body temperature, and reference electrodes were inserted subcutaneously in the forehead and above the tail. Goniovisc hypromellose 2.5% ophthalmic demulcent solution (Hub Pharmaceuticals, Rancho Cucamonga, CA) was applied to both eyes, and contact lens electrodes were then centered over left and right corneas, with Goniovisc acting as a fluid contact between each cornea and its electrode. Scotopic ERGs were recorded at six white light flash intensities ranging from −44 dB to 6 dB. EM software (LKC Technologies, Gaithersburg, MD) was used subsequently to calculate b-wave amplitudes [23, 24].

2.10. Histological examination

At PID 12 and PID 18, rats were sacrificed and the eyeballs were enucleated, fixed with 10% formalin, and embedded in paraffin by the Johns Hopkins University pathology lab. The paraffin sections were cut through the papillary optic nerve plane, and stained with hematoxylin and eosin (HE) for histological examination. Inflammation in the anterior chamber and the vitreous cavity was assessed. Retinal damage was evaluated through observation of retinal architecture. Sections were examined by a masked pathologist as follows [22, 25]: no tissue destruction, destruction of photoreceptor outer segment (light), destruction extending to outer nuclear layer (mild), destruction extending to inner nuclear layer (middle), and full thickness retinal damage (severe).

2.11. Real-time quantitative reverse transcription PCR (qRT-PCR)

The mRNA expression levels of inflammatory cytokines IL-17, IL-1β, and TNF-α were measured by qRT-PCR. Total RNA was extracted from the rat iris and retina at PID 12 and PID 18 by RNeasy mini kit (QIAGEN, Germany) according to manufacturer instructions. Total RNA concentration was determined using NanoDrop 2000 (Thermo Fisher Scientific, USA). cDNA was synthesized by reverse transcription of total RNA (1–2 μg) using a high-capacity cDNA reverse transcription system kit (Life Technologies, Carlsbad, CA). Inflammatory cytokines IL-17, IL-1β, and TNF-α sense primers and antisense primers were obtained from Integrated DNA Technologies (Coralville, IA). The sequences of primers are shown in Table S3 [26–28]. qRT-PCR was performed in triplicate for each sample using fast SYBR Green Master Mix (Life Technologies, Carlsbad, CA) and Step One Plus System (Life Technologies, Carlsbad, CA). The expression of target cytokines was normalized to the expression of GAPDH and calculated based on the comparative cycle threshold Ct method (2^−ΔΔCt).

2.12. Immunostaining and confocal imaging

The enucleated eyes from sacrificed rats were fixed in 2% paraformaldehyde phosphate buffer solution (PBS, 0.1M, pH 7.2) for 20 min after poking a hole in each cornea with a 30G needle. The cornea and iris were removed, followed by additional fixation for 90 min. After fixation and removal of the lens, tissues were rinsed with PBS containing 5% sucrose for 30 min and cryoprotected by consecutive immersion into 10% (30 min), 12.5% (30 min), 16.5% (30 min) and 20% sucrose in PBS at 4°C. The retinas were embedded in optimal cutting temperature (OCT) compound (Leica Microsystems, Buffalo Grove, IL), and serial cryosections with thickness of 20 μm were prepared and subsequently immunostained using the following antibodies and dyes: Iba1, 1:500, donkey anti-rabbit Cy2, and 4’,6-diamidino-2-phenylindole (DAPI) diluted in PBS containing 10% donkey serum and 0.1% Triton X-100. After overnight incubation in the primary antibodies at 4°C, the retina sections were washed three times in PBS and incubated with the secondary antibody at room temperature for 2 hours. The mounted retina sections were imaged using Zeiss LSM 710 confocal microscopy (Carl Zeiss, Germany). The number of Iba1-positive cells per image was automatically quantified using Fiji software (an open source imaging processing program from NIH), and the corresponding area in the retina was used to calculate cell density.

2.13. In vivo retina toxicity study of DSP-Zn-NP

DSP-Zn-NP (200 μg DSP per dose) were injected at one week intervals (total 4 doses) in healthy Lewis rats (n=6 eyes). Control eyes received SCT injection of saline (n=6 eyes) at the same interval and frequency. Retinal function was evaluated by recording scotopic ERGs at 1, 2, 3, and 4 weeks. At 4 weeks, rats were sacrificed after ERG recording and the eyeballs were enucleated for H&E staining and histological examination of the retinal structure.

2.14. Statistical analysis

Statistical analyses of EAU clinical scores were conducted by Two-way ANOVA followed by Tukey’s multiple comparison test. A one-tailed, unequal variance Student’s t-test was used to evaluate significance between two sets of data. *p<0.05; **p<0.01; ***p<0.001.

3. RESULTS

3.1. Trans-scleral transport of DSP compared to dexamethasone

Drugs administered by SCT injection are thought to penetrate into the eye by trans-scleral diffusion [29–31], a process that is limited for drugs that are poorly water-soluble [32–34]. We compared the penetration rates of water-soluble dexamethasone sodium phosphate, DSP, to poorly water-soluble dexamethasone in an ex vivo trans-scleral transport assay that utilized a Franz diffusion cell, wherein the donor and receptor compartments were separated by freshly dissected rabbit sclera with episclera facing the donor chamber [19]. We observed an 18-fold increase in trans-scleral penetration of DSP compared to dexamethasone at 6 h (Figure 1A). We then calculated the apparent scleral permeability coefficient (P), which is independent of time and surface area. The calculated P values were 19±3 ×10⁻⁶ cm/sec for DSP and 1.2±0.1×10⁻⁶ cm/sec for dexamethasone, a difference of >15-fold (Figure 1B).

Figure 1.

Ex vivo trans-scleral diffusion of DSP (0.1% DSP ophthalmic solution) and dexamethasone (0.1% Maxidex® ophthalmic suspension) with rabbit sclera. (A) Cumulative drug transport (μg) and (B) apparent permeability coefficients (P) for DSP and dexamethasone. Mean ± SEM (standard error of mean); Student’s t-test, ***p<0.001; n=3 per time point.

3.2. Preparation and characterization of DSP-Zn-NP

In order to load water-soluble DSP into biodegradable nanoparticles and achieve controlled DSP release for weeks, zinc was used to bridge the phosphate group of DSP with carboxyl-terminated PLGA with M_W of 5.6 kDa or 34 kDa. A drug loading of 6% w/w and 1.2% w/w was achieved for DSP-Zn-NP made using carboxyl-terminated PLGA5.6kDa and carboxyl-terminated PLGA34kDa, respectively (Table 1). In comparison, we were unable to detect DSP in the nanoparticles when ester-terminated PLGA (without terminal carboxyl group) was used (data not shown). Similarly, DSP content was below the limit of detection when zinc was not used with the carboxyl-terminated PLGA (Table 1). DSP-Zn-NP were spherical in shape (Figure 2A), with an average particle size of 210 ± 15 nm and 160 ± 8 nm for DSP-Zn-NP made using carboxyl-terminated PLGA5.6kDa and carboxyl-terminated PLGA34kDa, respectively (Table 1). DSP-Zn-NP prepared using F127 exhibited a nearly neutral surface charge of −9 ± 2 mV or −11 ± 5 mV (Table 1), indicating Pluronic F127 coated the NP surface [35]. In comparison, DSP-Zn-NP made without F127 possessed a zeta-potential of −64 mV (Table 1). F127 also reduced nanoparticle aggregation during centrifugation and washing steps and improved the overall NP yield. DSP-Zn-NP prepared using F127 showed narrow particle size distribution (Figure S1A and B) with small PDI <0.1 (Table 1), but without F127 the DSP-Zn-NP showed a high PDI of 0.38 (Table 1) indicating nanoparticle aggregation (Figure S1C). DSP was steadily released in vitro for up to 4 weeks from DSP-Zn-NP made using carboxyl-terminated PLGA5.6kDa and for more than 14 weeks from DSP-Zn-NP made using carboxyl-terminated PLGA34kDa (Figure 2B). DSP-Zn-NP made using carboxyl-terminated PLGA5.6kDa exhibited two phases of drug release, with an initial week of relatively fast drug release followed by relatively slow release for 3 weeks (Figure 2B insert); this formulation was selected for evaluation of pharmacokinetics and efficacy in animals.

Table 1.

Physiochemical characteristics of DSP-Zn-NP

DSP-Zn-NP	Coating	Zn2+	Diameter	PDI	Surface charge (mV)	Drug loading EE (%) (w/w %)	EE (%)
F127/PLGA5.6kDa	F127	+	210 ±15	0.10 ± 0.03	−9 ± 2	6.0	36
F127/PLGA5.6kDa	F127	−	110 ± 2	0.06 ± 0.01	−3 ± 2	0	0
PLGA5.6kDa	None	+	267 ± 57	0.38 ± 0.22	−64 ± 2	n/a	n/a
F127/PLGA34kDa	F127	+	160 ± 8	0.09 ± 0.01	−11 ± 5	1.2	7

PDI: polydispsersity index of particle size

EE: encapsulation efficiency

Figure 2.

(A) Representative TEM image and higher magnification inset of DSP-Zn-NP made using carboxyl-terminated PLGA5.6kDa and (B) the in vitro drug release profile of DSP-Zn-NP made using carboxyl-terminated PLGA5.6kDa or made using carboxyl-terminated PLGA34kDa. The insert shows expanded drug release profile of DSP-Zn-NP made using carboxyl-terminated PLGA5.6kDa. Mean ± standard deviation, n=3 repeats.

We further measured the ocular ³H-DSP levels in rats following a single SCT injection of either DSP solution or DSP-Zn-NP made using carboxyl-terminated PLGA5.6kDa (0.5 μCi or 100 μg DSP per eye, 30 μL injection). Nearly 65% of the total DSP dose was retained in the conjunctival space at 2 h after SCT injection of DSP-Zn-NP, and the amount of DSP in the conjunctival space gradually decreased to 5% and 0.05% of the initial dose by day 7 and day 21, respectively (Figure 3A). In comparison, when the DSP solution was administered by SCT injection (without NP formulation), only ~0.2% of the initial dose was retained at the injection site at 2 h after administration. DSP-Zn-NP provided significantly increased concentration of DSP in the aqueous humor (fluid in the anterior chamber between lens and cornea) and vitreous humor (fluid in the vitreous chamber between lens and retina) for up to 7 days, with a significant drop between day 7 and day 14 after injection (Figure 3B and3C). The concentration of DSP in the vitreous humor at day 7 and day 14 after SCT administration of DSP-Zn-NP was 0.44 ng/mg and 0.01 ng/mg, respectively. We also found that SCT administration of DSP-Zn-NP resulted in very low DSP levels in the blood (<0.05 ng/mg) at each time point measured (Figure 3D). In order to confirm that systemic absorption is not a significant route for intraocular drug penetration following SCT injection, we measured the DSP levels in an un-injected fellow eye after SCT DSP-Zn-NP injection. DSP levels in the aqueous humor and vitreous humor of the un-injected fellow eye were undetectable at 24 h post SCT injection of 100 μg DSP-Zn-NP in the opposite eye (Table 2).

Figure 3.

Pharmacokinetics of DSP and DSP-Zn-NP after SCT administration in rats. (A) Percentage of the initial DSP dose retained at the injection site, and DSP levels in the (B) aqueous humor, (C) vitreous humor and (D) blood. Mean ± SEM, n=4 eyes/group and n=2 blood samples/group. Student’s t-test. *p<0.05; **p<0.01; ***p<0.001.

Table 2.

Drug levels in un-injected fellow eye at 24 h after SCT injection of DSP-Zn-NP.

	Aqueous humor (ng DSP/mg)	Vitreous humor (ng DSP/mg)
Injected eye	3.53 ± 3.77	0.71 ± 0.13
Un-injected fellow eye	not detectable	not detectable

3.3. DSP-Zn-NP efficacy in EAU rat model

EAU was induced in Lewis rats by interphotoreceptor retinoid binding protein (IRBP) peptide, R16 (residues 1177–1191, sequence ADGSSWEGVGVVPDV) [18]. An inflammatory response with clinical score of 0.7±0.4 was observed on post-immunization day (PID) 8 (Figure 4A). EAU rats were treated with a single SCT injection of DSP-Zn-NP (containing 180 μg DSP), DSP (180 μg) or PBS at PID 8. The severity of EAU clinical score was significantly lower in the DSP-Zn-NP-treated group than those in the PBS- or DSP- treated groups from PID 9 until PID 18 (Figure 4A). The PBS-treated animals developed severe EAU at PID 12 with intensive pus accumulation in the anterior chamber (hypopyon) and inflammatory cell infiltration in the anterior segments of the eye, as shown in a representative slit lamp image (Figure 4B). Fundus photography of PBS-treated control animals exhibited cloudy vitreous with dilated retinal blood vessels in the posterior segments of the eye (Figure 4F). DSP treatment did not effectively control the disease either, as persistent hypopyon and inflammatory cell infiltration (Figure 4C), and cloudy vitreous (Figure 4G) were present in these animals. Strikingly, DSP-Zn-NP-treated rats only developed mild inflammation with mild inflammatory cell infiltration and no hypopyon (Figure 4D), which was evident in a clear fundus image (Figure 4H) that was similar to the healthy eye controls (Figure 4 E, I).

Figure 4.

Disease severity in EAU rats with different treatments. (A) Clinical observation of EAU rats treated with SCT injection of PBS, DSP and DSP-Zn-NP (arrow denotes the day of treatment). Slit lamp images (middle row) and Fundus images (bottom row) at post-immunization day (PID) 12 show ocular inflammation in (B, F) PBS treated groups and (C, G) DSP treated groups. In contrast, (D, H) DSP-Zn-NP treatment led to decreased ocular inflammation. (E, I) Healthy animals were used as control. Pus accumulation in the anterior chamber between cornea and lens (hypopyon, indicated as asterisks), inflammatory cell infiltration (arrows), and retinal blood vessel enlargement (triangles) were more evident and frequent in the groups that received PBS or soluble DSP. Mean ± SEM (n = 12–24 eyes). Two-way ANOVA followed by Tukey’s multiple comparison test. * p<0.05 PBS vs DSP; # p<0.05 PBS vs DSP-Zn-NP; ‡ p<0.05 DSP vs DSP-Zn-NP.

Histological examination showed DSP-Zn-NP-treated eyes had no damage to the retina, or damage only limited to the photoreceptor outer layer, while those treated with DSP and PBS showed retinal damage extending to the full retinal thickness. Inflammatory cell infiltration was evident in both the vitreous body (Figure 5A, E) and the anterior chamber (Figure 5H, L) for PBS-treated eyes at PID 12 and PID 18. Abnormal retinal folds were observed in PBS-treated eyes at PID 18 (Figure 5E). In comparison to PBS treatment, DSP treatment reduced inflammatory cell infiltration in both the vitreous (Figure 5B, F) and the anterior chamber (Figure 5I, M), but did not prevent the formation of abnormal retinal folds caused by severe retinal inflammation (Figure 5F), indicating retinal tissue damage in DSP-treated eyes. However, eyes treated with DSP-Zn-NP had minimal presence of inflammatory cells in either the vitreous (Figure 5C, G) or the anterior chamber (Figure 5J, N). The retinal architecture in DSP-Zn-NP-treated eyes was uniformly organized (Figure 5C, G), similar to that of healthy control eyes (Figure 5D).

Figure 5.

Histological findings of EAU rats with different treatments. The inflammation in EAU rats at both post-immunization day (PID) 12 and PID 18 was evident in groups of animals that received (A, E, H, L) PBS treatment and (B, F, I, M) DSP treatment, but was reduced in animals that received (C, G, J, N) DSP-Zn-NP treatment. (D, K) Healthy rats were used as control. Scale bar, 50 μm. Black arrows indicate the presence of inflammatory cells; black asterisks indicate the retinal folds; ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL).

3.4. Preservation of retinal function

To examine the ability of DSP-Zn-NP to preserve retinal function in EAU rats, we measured scotopic electroretinography (ERG) responses at PID 18, as shown in Figure 6. ERG measures the electrical response of the retina to light, and reduction of ERG amplitudes indicates functional changes in retinal cells due to retinal inflammation damage. PBS-treated control eyes showed the lowest amplitudes for scotopic b-wave, indicating the greatest destruction of retina function from EAU. Significantly greater b-wave amplitudes were observed in healthy, DSP-Zn-NP-treated, and DSP-treated eyes as compared to PBS-treated eyes at flash intensities of −34, −24, −14, −4, and 6 dB (p<0.05). Notably, DSP-Zn-NP-treated eyes showed significant improvement over eyes treated with DSP alone at flash intensities of −24, −14, −14, −4, and 6 dB (p<0.05). At 6 dB, there is a nearly 80% preservation of retinal function after DSP-Zn-NP treatment in comparison to the healthy control eyes and more than a 2-fold increase of the retinal function after DSP-Zn-NP treatment over the PBS-treated EAU eyes.

Figure 6.

ERG responses of EAU rats receiving different treatments. Mean scotopic b-wave amplitude at the study end point post-immunization day (PID) 18 was plotted against flash intensity. Representative p-values are shown at 6 dB. *p<0.05; **p<0.01; ***p<0.001, Student’s t-test. Mean ± SEM, n=6 eyes/treatment.

3.5. mRNA expression of various inflammatory cytokines

In EAU rats treated with PBS, the mRNA expression of IL-17, TNF-α, and IL-1β were significantly increased in both the retina and iris at PID 12 (Fig 7A) and PID 18 (Figure 7B). DSP-Zn-NP treatment significantly decreased the mRNA expression levels of IL-17, TNF-α and IL-1β at PID 12 in both retina and iris. DSP-Zn-NP were more effective than DSP in suppressing mRNA expression of IL-17 (p<0.001) and TNF-α (p<0.001) in retina, and IL-17 (p<0.001), TNF-α (p<0.001) and IL-1β (p<0.001) in the iris at PID 12. At PID 18, DSP-Zn-NP treatment resulted in mRNA expression levels for most cytokines that were similar to the healthy control, except IL-17 in the retina and TNF-α in the iris. In comparison, mRNA expression levels of IL-17, TNF-α, and IL-1β in the iris, and IL-17 in the retina with DSP treatment were still significantly higher than those of healthy controls at PID 18 (Figure 7).

Figure 7.

mRNA expression of inflammatory cytokines, expressed as a fold-change compared to healthy control eyes, at disease peak post-immunization day (PID) 12 and at the study end point, PID 18, in the (A) retina and (B) iris. *p<0.05; **p<0.01; ***p<0.001, Student’s t-test. Mean ± SEM, n=3 eyes/treatment.

3.6. DSP-Zn-NP treatment decreased microglial cell density in the retina

To further investigate the protective effect of DSP-Zn-NP in the retina, we characterized the activation and proliferation of microglia. The cell density of microglia in the retina was analyzed by immunostaining with antibodies against microglia marker Iba1 (ionized calcium-binding adapter molecule 1). The average cell density of microglia was 996±196 /mm² and 890±255 /mm² for PBS and DSP-treated retinas at PID 12, respectively (Figure 8A, B). DSP-Zn-NP treatment considerably reduced the density of microglia (285±43 /mm²) (Figure 8C). For comparison, a microglial density of 81±16 cells/mm² was measured in the healthy retina (Figure 8G). Moreover, the average microglial cell densities at PID 18 were 1494±200, 881±116, 292±26 cells per mm² in PBS, DSP, DSP-Zn-NP-treated retinas, respectively (Figure 8 D-F). At either time point, there was not a significant difference between healthy and DSP-Zn-NP-treated eyes (p=0.392 at PID 12 and 0.238 at PID 18).

Figure 8.

DSP-Zn-NP treatment decreased microglial density in the retina of EAU rats as compared to treatment with DSP or PBS. Retinas were immunostained with Iba1 (green) and DAPI (blue). The number of Iba1-positive cells in EAU retina was significantly decreased by DSP-Zn-NP treatment at (A-C) post-immunization day (PID) 12 and (D-F) PID 18. (G) Heathy retina was used as the control. (H) The cell density is expressed as mean ± SEM (n=3). Scale bar, 50 μm. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. *p<0.05; **p<0.01; ***p<0.001, Student’s t-test. Mean ± SEM, n=3 eyes/treatment.

3.7. DSP-Zn-NP treatment did not increase intraocular pressure (IOP)

We did not observe IOP increase in DSP-treated or DSP-Zn-NP-treated rats, as compared to PBS-treated rats, at any point during the 18-day follow-up (Figure 9).

Figure 9.

IOP measured on eyes of animals that received SCT injection of PBS, DSP-Zn-NP or DSP. Mean ± SEM, n=12–24 eyes/treatment. *p<0.05; **p<0.01; ***p<0.001, Student’s t-test.

3.8. SCT injection of DSP-Zn-NP do not cause retina toxicity

Four SCT doses of DSP-Zn-NP (200 μg DSP per dose), each spaced at one week intervals, did not cause any retinal toxicity in healthy rats, as assessed by histology and electroretinography (ERG). Histology confirmed that the retinal structure was not affected by weekly SCT injection of DSP-Zn-NP (Figure 10A), similar to saline control (Figure 10B). Furthermore, ERG scotopic b-wave and a-wave were not statistically different in rats that received 4 weekly injections of DSP-Zn-NP versus saline injections (Figure 10C and D).

Figure 10.

DSP-Zn-NP injections did not cause apparent retinal toxicity in healthy rats. Retinas appeared normal following 4 weekly SCT injections of (A) DSP-Zn-NP (200 μg DSP per dose) or (B) saline. ERG showed no reduction in (C) scotopic b-wave or (D) scotopic a-wave amplitudes over a range of intensities in the eyes injected with DSP-Zn-NP compared to those injected with saline. Scale bar, 50 μm. Mean ± SEM, n=6 eyes/treatment. *p<0.05; **p<0.01; ***p<0.001, Student’s t-test.

4. Discussion

Corticosteroids have been the mainstay of therapy for noninfectious uveitis, and corticosteroids may be administered either topically, systemically, or as periocular and intraocular injections. Topical corticosteroids are used to manage anterior uveitis, which only affects the front part of the eye. Intraocular (intravitreal) injections of corticosteroids directly to the vitreous body can treat posterior uveitis that affects posterior segments of the eye. Systemic corticosteroids to treat noninfectious uveitis are associated with undesirable systemic side effects [11]. Periocular (e.g. SCT injection) corticosteroids, which are injected along the eyeball as opposed to into it, can provide high, albeit short-lived, local concentrations of corticosteroids in both the anterior and posterior segments of the eye while eliminating or reducing systemic side effects. SCT corticosteroids are promising for treating panuveitis, which affects the whole eye, and posterior uveitis, which affects the back of the eye, but less invasive methods that provide prolonged duration of drug action are needed.

SCT delivery of ocular treatments has been utilized for decades, including corticosteroids for inflammatory disease and antibiotics for infectious disease. SCT injection is a promising method for delivery of controlled release medications [36–38]. Medications injected into the conjunctiva, a space composed of epithelia and connective tissue layers covering the sclera, do not penetrate the structural components of the eye. Thus, SCT injection obviates risks associated with intraocular injection, such as blurred vision, endophthalmitis, and retinal detachment. Although small, the risk of endophthalmitis and/or retinal detachment caused by intravitreal injection increases as patients receive an increasing number of intravitreal injections [39]. Furthermore, patients would likely prefer a less invasive SCT injection, especially if the delivery system is nanoparticles that can be administered with smaller needles [40], compared to the more invasive intravitreal injection.

Several possible routes have been proposed for effective intraocular drug penetration following the SCT administration [16, 41], including trans-corneal penetration [41], systemic absorption through the conjunctival vasculature [17, 42], and trans-scleral diffusion [29–31]. Trans-corneal penetration may be limited to times closely after the SCT injection, before the injection needle hole reseals, during which some drug may leak out and reach the corneal surface [41]. Systemic absorption of DSP following SCT injection can occur, as shown in the current study and previously [17, 42]. However, after SCT injection of DSP-Zn-NP, we did not detect DSP in the un-injected fellow eye, whereas the injected eye showed sustained DSP levels in both the aqueous and vitreous. Therefore, systemic absorption is unlikely to be the main route for intraocular penetration of DSP following SCT administration. On the other hand, we found that water-soluble DSP exhibited approximately 18-fold increased trans-scleral penetration through the rabbit sclera ex vivo as compared to water-insoluble dexamethasone. Scleral permeability varies by animal species and strain, and can be decreased by chronic experimental glaucoma [43]. The trans-scleral diffusion of a drug depends on its molecular weight, hydrophilicity and charge [34, 44]. Water-soluble small molecules typically show much higher trans-scleral drug penetration than hydrophobic drugs, perhaps due to the higher concentration gradients possible with more water-soluble molecules [10, 45]. Typically hydrophilic small molecules showed significant trans-scleral permeability [34, 44]. Thus, delivering water soluble DSP was essential to achieving high intraocular drug levels following SCT injection. However, the therapeutic effects of water-soluble corticosteroids injected SCT are short-lived [15], necessitating frequent injections [46]. Thus, a sustained release platform that can be administered less invasively by SCT injection to the eye, and that provides prolonged local release of corticosteroids, is expected to improve patient compliance and therapeutic efficacy for noninfectious uveitis.

In order to encapsulate high amounts of water-soluble DSP, we employed an ionic bridging method between carboxylic acid groups on carboxyl-terminated PLGA and the phosphate group on DSP [17]. A low molecular weight polymer was used to increase the carboxyl end group prevalence in a given weight of polymer since it was found to enhance drug encapsulation (presumably through zinc ion chelation with DSP). DSP-Zn-NP made using carboxyl-terminated PLGA3.2kDa exhibited a high drug loading of 8 wt%, but the majority of DSP was released within 1 week [17]. The ocular inflammation in EAU rat eyes occurs in ~1 week and peaks in ~2 weeks post-immunization, and the ocular inflammation lasts up to 1 month. A suitable treatment regimen for EAU would provide sustained high drug levels during the first week after the disease to quickly suppress the initial inflammation, followed by a prolonged period with low maintenance drug level to control the inflammation. In order to prolong drug release beyond 1 week, we utilized carboxyl-terminated PLGA with higher molecular weight (PLGA5.6kDa and PLGA34kDa). We optimized a DSP-Zn-NP formulation (DSP-Zn-NP made using carboxyl-terminated PLGA5.6kDa) that loaded 6% w/w of DSP and provided sustained levels of the corticosteroid over 4 weeks in vitro, with two release phases: an initial phase of fast drug release and a second phase of slow drug release. DSP-Zn-NP made using carboxyl-terminated PLGA34kDa provided more than 3 months of DSP release in vitro, but the drug loading was only 1% w/w, likely due to the lower content of carboxyl end groups available per gram of polymer resulting reduced ability to encapsulate DSP via zinc bridging. By formulating DSP-Zn-NP with a high loading and sustained release of water soluble DSP, we achieved significant levels of DSP over a prolonged period in both the anterior and posterior segments of the eye following SCT injection. With further modifications to the formulation, the potential for achieving drug release that is sustained for 6 months to 1 year upon SCT injection could be very attractive clinically.

DSP-Zn-NP were manufactured with components (e.g. Poloxamers and PLGA) that are classified as generally regarded as safe (GRAS) materials by the FDA. We achieved significant PEG coatings on DSP-Zn-NP, as determined by NP surface charge, by formulating DSP-Zn-NP by the nanoprecipitation-solvent diffusion method in the presence of Pluronic F127. Pluronic F127 is a triblock copolymer of polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO) family, which we found to provide dense PEG coatings on biodegradable NP compared to certain other pluronics, such as F68 [35]. We also previously discovered that dense PEG coatings on polymer NP, including PLGA NP, reduces ocular inflammation following injection into various parts of the eye, as compared to uncoated NP or NP coated with lower densities of PEG (e.g., coating with F68) [20]. PEG-coated DSP-Zn-NP made using carboxyl-terminated PLGA3.2kDa were found to be non-inflammatory after SCT injection in healthy rats [17]. Here, we found that 4 weekly injections of DSP-Zn-NP (PLGA5.6kDa) was similar to saline injections in the effects on retina structure and function. Key next steps in development include safety testing and pharmacokinetic analysis in larger animals, such as rabbits.

Noninfectious uveitis was previously correlated with presence of increased number of autoreactive CD4+ T cells in the eye, especially Th17 or Th1 cells [1, 47]. Hence, we investigated the expression of Th17 and Th1 related cytokines, including IL-17 and IL-1β. The SCT injection of DSP-Zn-NP significantly inhibited the mRNA expression of IL-17 and IL-1β in the eye. These findings suggest that the therapeutic effect of DSP-Zn-NP is associated with the inhibition of Th17 and Th1. Microglia respond rapidly to alterations of microenvironment in the retina and function to engulf pathogens [48]. Once activated, the microglia produce and release a variety of inflammatory cytokines, such as TNF-α under pathological conditions [49]. Corticosteroids can suppress the inflammatory response by inhibiting microglial activation and cytokine production [50, 51]. TNF-α is known to increase vascular endothelial permeability and lead to tissue damage [52, 53]. In the DSP-Zn-NP-treated eyes, significantly decreased mRNA expression of TNF-α may contribute to less intraocular inflammation and tissue damage. Iba1 plays an important role in the regulation of microglial function [54]. Iba1-positive cells may represent a subset of Cx3CR1 negative macrophages, but the majority of IbA-1-positive cells in retina are microglia [55]. Our results indicated that microglial cell density was significantly decreased by DSP-Zn-NP treatment when compared with PBS- or DSP-treatment, which was consistent with the downregulation of crucial inflammatory cytokines by DSP-Zn-NP. DSP may activate the glucocorticoid receptor and subsequently inhibit the self-activating loop in the microglia by suppressing cytokine production [56]. Our study further suggests that, in contrast to the rapid clearance of free drugs, the sustained release of DSP by nanoparticles maintains effective drug concentrations to suppress microglial activation and consequently downregulate multiple inflammatory cytokines.

The EAU model is an acute autoimmune uveitis model, with intraocular inflammation lasting for ~3–4 weeks [21]. In this study, we started the treatment 8 days after the EAU induction (PID 8) when mild/moderate intraocular inflammation was already underway. The peak of inflammation occurred by day 10, after which the clinical score slowly declined in the PBS control group. Injection of both DSP-Zn-NP and free DSP provided a reduction in the peak clinical score at PID 10, though only the sustained release from the DSP-Zn-NP led to a significant additional reduction in resolution of inflammation from PID 11 to PID 17. Further, the added reduction in inflammation provided by DSP-Zn-NP led to improved preservation of retinal function compared to free DSP in this acute model. We anticipate that the benefit of sustained DSP release would be even more apparent in chronic autoimmune uveitis models, such as R161H and AIRE^−/−, that last for ~4 months [57, 58], which is a topic for future study.

5. Conclusions

SCT administration of biodegradable nanoparticles loaded with dexamethasone sodium phosphate (DSP-Zn-NP) provided prolonged effective DSP concentrations in the aqueous and the vitreous of rats. Compared with the SCT injection of DSP solution, a single SCT injection of DSP-Zn-NP effectively treated EAU in rats, evidenced by significantly lower clinical scores, decreased mRNA expression of cytokines, less damage to the retina with reduced inflammatory cell infiltration, and preserved retinal function. The sustained DSP levels in both the vitreous and the aqueous for 2 weeks following the SCT injection of DSP-Zn-NP effectively diminished ocular inflammation and protected retinal function. SCT injection of DSP-Zn-NP presents a feasible and promising therapy for chronic noninfectious uveitis. The treatment may improve therapeutic outcomes by increasing efficacy and enhancing patient compliance. This work provides proof-of-concept insights into developing NP formulations that are suitable to be administered through less invasive SCT injection as a treatment option for noninfectious uveitis.

Highlights.

Single subconjunctival DSP-Zn-NP effectively treated experimental autoimmune uveitis

Carboxyl-terminated PLGA achieved high DSP loading in PLGA nanoparticles

Water soluble DSP is critical for effective trans-scleral drug transport

DSP-Zn-NP significantly reduced microglia cell density in the retina of EAU rats