Cyclosporine A-Loaded Nanocarriers for Topical Treatment of Murine

Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Cyclosporine A‑Loaded Nanocarriers for Topical Treatment of Murine Experimental Autoimmune Uveoretinitis Maren Kasper,*,† Doris Gabriel,‡ Michael Möller,§ Dirk Bauer,† Lena Wildschütz,† Herve Courthion,‡ Marta Rodriguez-Aller,§ Martin Busch,† Michael R.R. Böhm,∥,⊥ Karin Loser,# Solon Thanos,⊥ Robert Gurny,‡,§ and Arnd Heiligenhaus†,∇ †

Department of Ophthalmology and Ophtha-Lab, St. Franziskus Hospital, Münster 48145, Germany Apidel SA, Geneva 1201, Switzerland § School of Pharmaceutical Sciences, University of Geneva and University of Lausanne, Geneva 1221, Switzerland ∥ Department of Ophthalmology, Clinic for Diseases of the Anterior Segments of the Eyes, Essen University Hospital, Essen 45147, Germany ⊥ Institute for Experimental Ophthalmology and #Department of Dermatology, University of Münster, Münster 48149, Germany ∇ University of Duisburg-Essen, Essen 47057, Germany

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ABSTRACT: In the present study, tissue distribution and the therapeutic effect of topically applied cyclosporine A (CsA)loaded methoxy-poly(ethylene-glycol)-hexyl substituted poly(lactic acid) (mPEGhexPLA) nanocarriers (ApidSOL) on experimental autoimmune uveitis (EAU) were investigated. The CsA-loaded mPEGhexPLA nanocarrier was tolerated well locally and showed no signs of immediate toxicity after repeated topical application in mice with EAU. Upon unilateral CsA treatment, CsA accumulated predominantly in the corneal and sclerachoroidal tissue of the treated eye and in lymph nodes (LN). This regimen reduced EAU severity in treated eyes compared to PBS-treated controls. This improvement was accompanied by reduced T-cell count, T-cell proliferation, and IL-2 secretion of cells from ipsilateral LN. In conclusion, topical treatment with CsA-loaded mPEGhexPLA nanocarriers significantly improves the outcome of EAU. KEYWORDS: drug delivery, cyclosporine, nanocarrier, micelles, T-cells, uveitis, ophthalmics

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INTRODUCTION Cyclosporine A (CsA) is a lipophilic, poorly soluble, cyclic polypeptide routinely used as a systemic immunosuppressive drug after solid organ transplantations. Thereby, CsA facilitates a selective suppression of T-cells by inhibiting the calcineurin pathway, leading to cell cycle arrest and downregulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB)-induced proinflammatory gene expression (e.g., IL2).1,2 In addition to efficacy in transplant medicine, systemic CsA therapy has been used to treat various T-cell-mediated ocular inflammatory disorders, for example, dry eye syndrome,3 graft rejection after keratoplasty,4,5 and noninfectious posterior uveitis.6 Noninfectious intermediate and posterior uveitis encompass various immune-mediated diseases affecting the inner structures of the eye.7,8 Posterior uveoretinitis may lead to progressive loss of vision and reduce quality of life.9−11 The therapeutic efficacy of corticosteroids has been proven, and they constitute the backbone of uveitis treatment.12,13 However, in case of adverse effects or for managing refractory cases, systemic treatment with immunosuppressive agents has shown beneficial effects.13,14 Various immunosuppressive © XXXX American Chemical Society

agents are used, e.g., antimetabolites and alkylating agents, and T-cell inhibitors such as the calcineurin inhibitor CsA are the mainstay of therapy.13 Although systemic CsA is commonly used in uveitis therapy, long-term treatment comprises an increased risk of nephrotoxicity and hepatotoxicity.6 Thus, topically applied CsA eye drops represent an attractive strategy to achieve local immunosuppression with minimal systemic side effects in these patients. In contrast to systemic CsA therapy, a formulation for topical treatment of ocular disease must meet other requirements to reach a therapeutic level of 50−300 ng of CsA/g of tissue.15 The ideal formulation should be well tolerated, easily administered, and show an increased residence time on the cornea but have low systemic absorption to avoid systemic side effects and enable the drug to enter the eye in order to reach a therapeutic level at the site of inflammation. To achieve this goal, several ocular tissue Received: January 9, 2018 Revised: May 30, 2018 Accepted: June 4, 2018

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DOI: 10.1021/acs.molpharmaceut.8b00014 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

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barriers, such as the corneal and conjunctival epithelium as well as the blood−aqueous and blood−retina barriers need to be overcome.16 However, more than 90% of administered eye drops are absorbed in the systemic blood circulation via conjunctival and nasal blood vessels, while less than 5% enter the eye via corneal tissue.16 Therefore, they must be applied frequently to reach therapeutic levels in the anterior part, whereby drug concentrations are not adequate in the posterior part of the eye. CsA is a hydrophobic drug with very low water solubility, bioavailability, and corneal permeability. New developments in the topical delivery of CsA are focused on drug-impregnated contact lenses to prolong the precorneal residence time and in order to improve drug absorbance in the cornea by chemically modifying the drug (prodrug) or employing new delivery systems (e.g., emulsions, ointments, or liposomes).17 However, the hydrophobic characteristics and poor water solubility of CsA limit the development of an aqueous formulation.17,18 A milky emulsion-based formulation of CsA (Restasis 0.05%, Allergan Inc., Irvine, CA) is available for dry eye syndrome, but the capacity of this formulation limits the final CsA concentration to 0.5 mg/mL.19−21 By encapsulating CsA into mPEGhexPLA nanocarriers, the drug can be efficiently solubilized at a 10× higher concentration (5 mg/mL) and yields a perfectly transparent colloidal aqueous solution (0.5% CsA-ApidSOL). Incorporation of CsA in mPEGhexPLA polymeric micelles using amphiphilic copolymers has the advantage that the hydrophobic core protects the drug, while the hydrophilic corona stabilizes the micelles in the aqueous environment. Thereby, tissue penetration of CsA can be improved.22 Furthermore, polymeric nanocarriers have the advantage to escape the uptake by monocytes and the renal clearance, thereby allowing a prolonged circulation time in the body.23 In addition, CsA displays a high binding affinity to erythrocytes in the blood; 24 the outer shell of the mPEGhexPLA nanocarrier consists of a hydrophilic polymer that minimizes the interaction with blood components and displays no hemolytic capacity, thereby prolonging the circulation time in the body22 These mPEGhexPLA-based micellar nanocarrier formulations have been previously demonstrated to be nontoxic and well tolerated and to efficiently deliver poorly soluble drugs to the anterior and posterior compartments of the eye in rats.22,25−30 Small size and inert surface properties of mPEGhexPLA nanocarriers facilitate CsA delivery across biological barriers after local application to the eye. The depot formation within the ocular tissue mainly in the cornea facilitates enhanced drug levels in the anterior eye compartment. The penetration by the trans-scleral absorption route to the posterior part of the eye leads to enhanced levels in the retina as shown in keratoplastic model in rats.29 Good therapeutic efficacy in anterior eye diseases has been shown previously; analysis of the therapeutic efficacy of CsA-loaded mPEGhexPLA formulation in ocular diseases affecting the posterior part of the eye (e.g., autoimmune posterior uveitis) would be of great interest. The experimental autoimmune uveoretinitis (EAU) model in mice is a T-cell mediated autoimmune disease displaying pathologic features reminiscent of the pathology seen in patients with noninfectious endogenous uveitis.31 In the present study, we analyzed the tissue distribution and the therapeutic efficacy of topically applied CsA-loaded mPEGhexPLA nanocarriers on EAU in B10.RIII mice.

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MATERIAL AND METHODS

Preparation and Characterization of the mPEGhexPLA Formulation. Methoxy-poly(ethylene-glycol)hexyl-substituted poly(lactic acid) (mPEGhexPLA) is a pegylated poly(2-hydroxyoctanoic acid) polymer, forming spontaneously nanosized micelles in water (ApidSOL, provided by Apidel SA, Geneva, Switzerland). CsA was purchased from LC laboratories (Woburn, USA). Amicon ultra MW cutoff 3 kDa filter units were purchased from Millipore (Billerica, USA). Sterile water and a sterile PVDF 0.22 μm filter were purchased from Brunschwig (Basel, Switzerland). CsA/mPEGhexPLA micelle formulations were prepared at a 10 mL scale. Briefly, 500 mg of mPEGhexPLA copolymer and 50 mg of CsA were dissolved in 1 mL of acetone. The organic phase was then added to 10 mL of 10 mM phosphate buffer under sonication (Branson, 20% amplitude). Formulations were characterized in terms of size, morphology, and drug loading, using dynamic laser scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments, UK), transmission electron microscopy (TEM) (FEI Tecnai G2 Sphera, USA), and high-pressure liquid chromatography (HPLC) (Agilent, USA), respectively.28,32 Mice. B10.RIII mice are highly susceptible to developing EAU and possess a well characterized disease course. This model allows the analysis of new therapeutic strategies with a systemic, topical, or intraocular approach.31,33−36 B10.RIII mice (8 weeks of age, female) were purchased from Jackson Laboratory (Bar Harbor, USA). Animals were housed in standard animal rooms under a 12 h light/dark cycle with food and water provided ad libitum. The animal experiments were performed in accordance with the European Health Law of the Federation of Laboratory Animal Science Associations (FELASA) and the German Regulation of the Society for Laboratory Animal Science (GV-SOLAS). The protocol was approved by the North Rhine-Westphalia State Agency for Nature, Environment, and Consumer Protection (LANUV) (authorization number 84−02.04.2012.A044). All experimental procedures conformed to the guidelines of the National Institutes of Health and to the Association for Research in Vision and Ophthalmology resolution on the use of animals in research.33 Reagents. Human interphotoreceptor retinoid-binding protein peptide 161−180 (hIRBPp161−180) was purchased from the EMC Microcollection (Tuebingen, Germany). Incomplete Freund́s adjuvant (IFA) was obtained from Sigma-Aldrich (Steinheim, Germany). Heat-killed H37Ra mycobacteria tuberculosis was obtained from Invivogen (Toulouse, France). Immunization and Organ Collection. Mice were immunized (day 0) with 100 μg of hIRBPp161−180 in 0.1 mL of emulsion 1:1 vol/vol with complete Freundś adjuvant (CFA; IFA containing 2.5 mg/mL of H37RA). The emulsion was injected subcutaneously at the base of the tail.36,37 Mice were sacrificed on day 21 postimmunization (p. i.), and the cervical and inguinal LN, spleen, brain, liver, kidney, peripheral blood, and eyes were collected for further analysis. Ocular Treatment. To obtain a therapeutic approach, topical treatment started at day 12 after EAU induction to ensure an established ocular inflammation. On days 12−21 p. i., the right eyes from mice received 10 μL of CsA/ mPEGhexPLA micelle formulation (n = 18) or PBS (n = 18) as a control five times a day at 3 h intervals. 10 μL of 0.5% CSA/mPEGhexPLA micelle formulation contained 0.05 mg of B


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Figure 1. CsA tissue distribution. The accumulation of CsA in (A) ocular tissues COR L (cornea left eye, n = 8), COR R (cornea right eye, n = 7), RET L (retina left eye, n = 8), RET R (retina right eye, n = 7), CHO L (sclera-choroid left eye, n = 8), CHO R (sclera-choroid right eye, n = 8) and in (B) peripheral organs LN L (cervical contralateral LN n = 8), LN R (cervical ipsilateral LN right, n = 8), LN I (inguinal lymph nodes, n = 8), LU (lung, n = 7), LI (liver, n = 8), SP (spleen, n = 8), BR (brain, n = 8), and (C) sera (sera, n = 7) was analyzed via UPLC/MS. The values for the CsA-treated group are shown after subtracting background values of the PBS-treated group. Bars indicate the mean ng of CsA/mg of tissue or ng of CsA/mL of sera (Kruskal−Wallis test * p < 0.05).

Specimens were stained with hematoxylin and eosin (HE) for histological examination. EAU severity was evaluated in a masked fashion on a scale of 0−4 using criteria based on number, type, and size of lesions as previously described.33,37 Flow Cytometry. Isolated cells from spleen or LN were separately stained with anti-CD4 FITC and anti-CD3 PE (eBioscience, Frankfurt a. Main, Germany) according to the manufacturer’s instructions. The cells were analyzed via flow cytometry (BD FACS Callibur). At least 5000 CD3+ events per sample were counted using CellQuest software (BD, Heidelberg, Germany) and analyzed using Cytomation Summit software (Dako). Lymphocyte Proliferation Assay and Cytokine Detection. Cells isolated from cervical LN were stained separately with the proliferation dye efluor 647 (eBioscience, Frankfurt a. Main, Germany), cultured in 96-well, roundbottom plates (1 × 105 cells/well), and stimulated with 50 μg/ mL of hIRBPp161−180, ConA (positive control) or medium (negative control). Cells were cultured in RPMI 1640 (Biochrom, Berlin, Germany), supplemented with 5 mM HEPES (Sigma-Aldrich), 5 × 10−4 M β-mercaptoethanol (Sigma-Aldrich), 10% fetal calf serum (Biochrom, Berlin, Germany), and 10 000 units/mL of penicillin and 10 mg/mL of streptomycin (Sigma-Aldrich).36 After 72 h of incubation, the cells were stained with anti-CD4-FITC, anti-CD3-PE, and 7AAD (eBioscience) according to the manufacturer’s instructions. The cells were then analyzed via flow cytometry to assess the proliferation of CD3+CD4+7AAD- T-cells (BD FACSCalibur). At least 5000 CD3+CD4+7AAD- events per sample were counted using the CellQuest software (BD, Heidelberg, Germany) and analyzed using Cytomation

CsA; the total administered drug dosage per mice averaged 0.25 mg of CsA/day (15.6 mg of CsA/kg/day). Because of the reconstitution of drug-loaded micelles in phosphate buffered solution and according to previous studies of topical regimen,29,30,34,38−42 PBS was used as control treatment in this study. CsA Dosage of Mice. Mice were sacrificed on day 21 p. i., and the cervical/inguinal LN, spleen, brain, liver, kidney, cornea, retina, sclera-choroid, and peripheral blood for preparation of sera samples of PBS- (n = 8) and CsA-treated mice (n = 8) were collected and stored at −80 °C until further analysis. CsA was quantified in collected samples as previously described.29 In brief, the tissues were thawed at room temperature, weighted, manually grinded, and then treated as follows: 400 μL of methanol, containing 100 ppb of deuterated CsA as internal standard, was added to either serum or tissue of interest (spleen, brain, liver, kidney, and cervical and inguinal LN samples). A volume of 200 μL of methanol was added to the cornea, retina, and choroid samples. The tissues were left under stirring/vortexing for 12 h. Afterward, the samples were centrifuged, and 150 μL of the supernatant was removed and transferred to UPLC (ultra performance liquid chromatography) vials. Subsequently, the CsA amounts were quantified with UPLC and following mass spectrometry (Water, USA) as described previously.43 The limit of quantification (LOQ) and limit of detection (LOD) were determined to be 2 ng/mL (ocular tissue), 0.4 ng/mL (organ tissue), and 0.32 ng/mL (sera), respectively. Evaluation of EAU. Paraffin-embedded eyes were cut in serial sections of 7 μm in a mediosagittal orientation. C


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Figure 2. Histopathological EAU-score is reduced by topical CsA/mPEGhexPLA treatment. Histopathological EAU-score was determined via HE staining, e.g., (A) EAU-score 0, (B) EAU-score 2. (C) The EAU severity from the ipsilateral, right (R) sham-treated (PBS, n = 10) and CsA/ mPEGhexPLA-treated (CsA, n = 10) eye and the contralateral, left (L) untreated eye of mice were assessed on day 21 after EAU induction (Kruskal−Wallis test * p < 0.05; comparison of right eyes Mann−Whitney U test * p < 0.05). (D) EAU severity of all eyes from the PBS (n = 20) and CsA/mPEGhexPLA-treated (n = 20) mice (Mann−Whitney U test * p < 0.05). Bars indicate the mean EAU-score of each group.

Compared to the nontreated eye, a significantly enhanced amount of CsA was found in corneal and sclera-choroidal tissue of the treated eye (p < 0.05; Figure 1A). In contrast, retinal tissues showed a significantly lower concentration of CsA (p < 0.05, Figure 1A) without demonstrating a statistically significant difference between the treated and the nontreated eye. Analysis of peripheral organs showed a striking accumulation of CsA (ng/mg) in lymphoid tissues, particularly in the cervical ipsi (0.6 ± 0.2) and contralateral LN (0.5 ± 0.1) as well as the inguinal LN (0.5 ± 0.1; Figure 1B). In comparison to the LN, reduced concentrations of CsA were detected in the kidney (0.29 ± 0.13), liver (0.23 ± 0.08), lung (0.12 ± 0.28), spleen (0.22 ± 0.08), and brain (0.02 ± 0.02) of the CsA-treated mice (Figure 1B). Finally, CsA was detected in the sera of CsA/mPEGhexPLA-treated mice (0.47 ± 0.25 ng/mL; Figure 1C). Improvement in EAU after Topical Treatment with CsA/mPEGhexPLA. Histopathological evaluation of the eye sections revealed an EAU incidence of 100% in PBS- and CsA/ mPEGhexPLA-treated mice. In order to measure the CsA specific effect, EAU-scores including all samples of the PBS (n = 20; 1.3 ± 0.2) and CsA group (n = 20; 0.8 ± 0.1) were statistically analyzed. Therein, a significant reduced EAU-score in CsA/mPEGhexPLA-treated group was shown (p < 0.05; Figure 2D). The analysis of the single treated and nontreated eyes showed a mean severity score of 1.1 ± 0.2 in the PBStreated (right) eyes and 1.5 ± 0.3 in the nontreated (left) eyes (n = 10; Figure 2C). Disease severity of CsA/mPEGhexPLAtreated mice eyes was 0.7 ± 0.2 (n = 10; p < 0.05; Figure 2C) and was not reduced in comparison to nontreated eyes (1.0 ±

Summit software (Dako). The 72 h cell culture supernatants were harvested and analyzed for their IL-2 content by ELISA (Biolegend, London, UK) according to the manufacturer’s instructions. Data Analysis. According the CsA dosage analysis of mice, the results of one experiment are shown. With regard to the EAU-score, T-cell frequency, proliferation, and ELISA, the results of one representative experimental setting out of two with similar results are shown in the current manuscript. Data are displayed as mean value and standard error of mean (SEM). The data were processed with Med Calc V10 (MedCalc Software bvba, Belgium). The normal distribution was analyzed by the D’Agostino−Pearson test. Histopathological findings were analyzed by the Kruskal−Wallis Test and Mann−Whitney U test. The results of the flow cytometry and cytokine ELISA were analyzed by the Kruskal−Wallis Test. p < 0.05 was considered as a statistically significant difference between the groups.

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RESULTS Tissue Distribution of CsA/mPEGhexPLA after Topical Application. Mice received unilateral therapeutic application of CsA/mPEGhexPLA eye drops in the right eye starting at day 12 p. i. Thereby, 10 μL of the formulation or control item was applied five times per day to the right eyes. To assess the tissue distribution of CsA after unilateral treatment with CsA/ mPEGhexPLA eye drops, ng of CsA/mg of tissue of cornea, retina, sclera-choroid, cervical LN, inguinal LN, lung, liver, kidney, spleen, and brain and ng of CsA/mL of sera were analyzed via UPLC/MS (Figure 1). D


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Molecular Pharmaceutics 0.2; Figure 2C) When comparing the right treated eyes of the PBS and CsA/mPEGhexPLA group, no significant difference was shown (p = 0.1431). Influence of Topical CsA/mPEGhexPLA on Systemic Immune Responses in EAU. To assess the immunosuppressive effect of topical CsA/mPEGhexPLA treatment on Tcells, the frequency of CD3+CD4+ T-cells was analyzed by flow cytometry. Thereby, fewer CD3+CD4+ T-cells were found in the ipsilateral LN (11.8 ± 2.2) than in the contralateral (19.2 ± 1.2) LN of the CsA-treated group than determined in the ipsilateral (17.6 ± 2.3) and contralateral LN (18.4 ± 1.8) of the PBS-treated group (p < 0.05; Figure 3A).

Figure 4. CsA treatment reduces T-cell proliferation. Lymphocytes from cervical left (contralateral) and right (ipsilateral) LN (LN L; LN R), from sham- (PBS, n = 10) and CsA/mPEGhexPLA-treated (CsA, n = 10) mice were isolated and stimulated with medium, IRBP, or ConA. The ΔIRBP- and ΔConA-induced proliferative responses of CD3+CD4+ T-cells were determined by efluor 647 staining. Data are averages of one experiment. Bars indicate the mean score of each group (Kruskal−Wallis test * p < 0.05).

Figure 3. Topical CsA treatment affected T-cell population. Frequency of CD3+CD4+ and T-cells of lymphocytes from cervical left (contralateral), right (ipsilateral), and inguinal LN (LN L; LN R; LN I) and spleens from sham-treated (PBS, n = 10) and CsA/ mPEGhexPLA-treated (CsA, n = 10) mice were determined. Data are averages of one experiment. Bars indicate the mean score of each group (Kruskal−Wallis test * p < 0.05).

Neither contralateral and inguinal LN (PBS: 19.2 ± 2.2; CsA: 22.0 ± 2.9) nor splenic (PBS: 22.4 ± 3.5; CsA: 27.7 ± 2.9) CD3+CD4+ T-cells were altered due to the unilateral topical CsA/mPEGhexPLA treatment, and no significant differences within the PBS group were detected (Figure 3). Since the frequency of T-cells in cervical LN was reduced in the CsAtreated mice, we analyzed the characteristics of the T-cells in more detail. CsA/mPEGhexPLA treatment did not lead to a significantly reduced IRBP-induced proliferation from cells of ipsi (4.8 ± 1.8) and contralateral (6.5 ± 3.6) LN compared to that of the control group (ipsi 8.8 ± 2.3; contra 5.9 ± 2.5; Figure 4). Only mitogen-induced proliferation was significantly reduced from T-cells of the ipsilateral LN (17.3 ± 5.6) as compared to the contralateral LN (33.6 ± 5.1) of the CsAtreated group (Figure 4; p < 0.05). In concordance with the decreased number of T-cells, cells from ipsilateral cervical LN of CsA/mPEGhexPLA-treated mice contained a significantly lower IRBP-induced secretion of IL-2 (1.2 ± 1) compared to contralateral LN (13.6 ± 4.1; Figure 5, p < 0.05). Furthermore, IRBP-induced IL-2 secretion was significantly lower in cervical ipsilateral LN than in the control group (ipsi 7.3 ± 2.0, p < 0.05; contr 3.6 ± 1.3; Figure 5). Finally, mitogen-induced IL-2 secretion decreased significantly in the ipsilateral LN (54.8 ± 22.4) as compared to

Figure 5. CsA treatment affected the IL-2 secretion. Lymphocytes from cervical left (contralateral), right (ipsilateral), and inguinal LN (LN L; LN R) from sham- (PBS, n = 10) and CsA/mPEGhexPLAtreated (CsA, n = 10) mice were isolated and stimulated with medium, IRBP, or ConA. ΔIRBP- and ΔConA-induced cytokine secretion (pg/mL) of IL-2 was determined by ELISA. Data are averages of one experiment. Bars indicate the mean score of each group (Kruskal−Wallis test * p < 0.05).

the contralateral LN (94.0 ± 25.3) of the CsA/mPEGhexPLAtreated and to the cervical LN of the PBS-treated mice (ipsi 152 ± 16.6; contra 140.9 ± 22.6; Figure 5; p < 0.05).

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DISCUSSION With regard to the development of effective noninvasive intraocular drug delivery systems, polymeric micelles are one of the most promising concepts for the management of ocular E


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commonly known to circulate in the lymphatic system.54 Therefore, in addition to local effects, CsA-loaded mPEGhexPLA nanocarriers exerts a systemic immunosuppressive effect after topical administration in EAU. Overall therapeutic CsA levels of at least 50−300 ng of CsA/ g15 were achieved in the majority of ocular and peripheral tissues. Furthermore, CsA was detected in sera but only at low concentrations below 2 ng/mL so that typical CsA side effects are not expected.55,56 The systemic detection in solid organs and serum might be due to the extensive absorption of eye drops via conjunctival and nasal blood vessels.16 Similar to ours and previous distribution studies by Di Tommaso et al.,29,30 topical treatment with CsA-loaded mPEG−PLA nanocarrier results in an enhanced corneal accumulation and enhanced CsA level in the anterior chamber of rabbits.57 In comparison to standard mPEG−PLA nanocarrier, it has been shown that the mPEGhexPLA nanocarrier possess an increased hydrophobicity of the micellelar core, which enables higher drug loading.58 In the current study, PBS was used as a control and leads to an unexpected slight reduction of the EAU severity of the treated eyes. Other groups have shown that changes in the salt concentrations in vitro and in vivo may lead to a modulation of immune cells. NaCl led to an induction of Th17 cells and to an exacerbation of autoimmunity.59 In contrast, KCl led to an inhibition of Th17 cells in vitro.60 We did not find a significantly reduced immune response in draining lymph nodes of PBS-treated mice, while EAU-scores from PBStreated eyes have been reduced. Further, the EAU-score of PBS-treated eyes never reached a level of significance. Therefore, we could not find a therapeutic effect of topical PBS treatment in EAU. Several studies showed that systemically applied 10−20 mg of nCsA/kg/day efficiently prevented disease development in rodent EAU models.6 Furthermore, Nussenblatt et al. showed the prophylactic efficacy of topical bilateral treatment with 0.5% CsA eye drops in the EAU rat model, which was associated with an elevated CsA level in sera (285 ng/mL).61 In contrast, therapeutic bilateral (0.5% CsA) or unilateral (2% CsA) topical treatment was ineffective in EAU.61 The decreased therapeutic efficacy of topical installation has been reasoned by the lack of drug penetration through the corneal tissue into the eye and a limited systemic effect reflected by the low CsA level in sera.61 An improved ocular absorption of CsA has been shown for the CsA-loaded mPEGhexPLA nanocarrier in rabbits and rats accompanied by a low serum level of CsA.29,30 Topical treatment with the CsA-loaded mPEGhexPLA nanocarrier in an experimental model of ulcerative colitis lead to increased local concentrations but low serum level.62 In our study, 0.5% CsA (15.6 mg of CsA/kg/day) has been used for a unilateral therapeutic regimen in EAU leading to an improvement in the treated eyes without an elevated CsA level in serum. However, elevated concentrations of CsA in the corneal and sclera-choroidal tissue in the current study and the low serum level are in line with the study of Nussenblatt et al., who also described an accumulation of CsA in the corneal and posterior scleral tissue61 and lead us to the suggestion of a local immunosuppressive mechanisms of this topical treatment regimen. The therapeutic efficacy of the local treatment was consistent with the levels of CsA found in the cornea and the sclera-choroidal tissue of the treated eyes. As shown for rats, lower CsA levels were detected in the retinal tissue.29

diseases affecting the intraocular anterior and posterior segments of the eye.44 Polymeric micelles are highly effective delivery systems for intraocular drug delivery, facilitating the drug administration of hydrophobic drugs, enabling constant drug release, and improving local therapeutic effects, while unwanted systemic side effects are reduced.44 The nontoxicity of empty and CsA-loaded mPEGhexPLA micelles has been shown earlier in vitro and vivo.22,25−30 Topical administration of CsA/mPEGhexPLA micelles are shown to overcome ocular barriers and to provide therapeutic level in anterior and posterior segments of the eye, being effectively therapeutic in comparison to PBS or CsA/oil formulation.29,30 In the present study, the unilateral therapeutic treatment using a novel aqueous-based CsA-loaded mPEGhexPLA-based nanocarrier formulation improved EAU severity in mice. In the current study, we investigated the tissue distribution and the effector mechanisms behind the observed therapeutic effect of locally delivered CsA on EAU. In agreement with previous studies,22,27−29,36 our data document a good systemic and local tolerance of topical CsA/mPEGhexPLA after repeated (5×/ day) application to the eye for nine consecutive days. No local adverse effects in eyes and periocular tissues (e.g., blepharitis) or systemic side effects (e.g., weight loss) of CsA/ mPEGhexPLA-treated mice were observed (data not shown). Topically applied drugs enter the eye by both a corneal and a noncorneal (conjunctiva, sclera) pathway.45 In the current study, quantification of CsA in the collected ocular and peripheral tissues showed a higher accumulation of CsA in the cornea of the treated eye and to a lesser extent in the nontreated eye. This has also been shown in unilateral ocular topical administration study of spironolactone-loaded mPEG-dihexPLA micelles in rabbits.46 This observation has been explained by known interconnections between the eyes facilitating a direct transport of the drug to the contralateral eye. Return of the drug to the contralateral eye through the general circulation or more unlikely by the grooming behavior of the mice after each application might explain this observation.46 The enhanced concentration of CsA in the corneal tissues of the treated eyes is in line with previous ocular distribution studies of CsA/mPEGhexPLA formulations in a keratoplastic rat model and indicate that the corneal absorption route is efficient, leading to formation of a corneal depot and enhanced drug level in the anterior parts of the eye. In addition, elevated retinal CsA levels were shown, leading back to a trans-scleral absorption route.29 CsA content was very low in retinal tissue in the current study, with slightly elevated CsA concentrations in the sclerachoroidal tissue of the treated eyes. The very low intraocular concentration of CsA shown here in the retinal tissue might be explained by the aqueous humor dynamics of the eye and the fast clearance time for intraocular drugs via the ocular lymphatic drainage and uveoscleral outflow.47−50 In addition to the corneal and sclera-choroidal tissue of the treated eyes, CsA was measured in the cervical ipsi and contralateral as well as inguinal LN, in the spleen, kidney, and liver. CsA accumulation in cervical ipsilateral LN might be related to the uveolymphatic pathway between the eye and draining cervical LN shown in mice.48−53 Thereby, the nanocarriers might enter the lymphatic system and distribute to neighboring and distant LN. From drug delivery systems for tumor metastasis, biodegradable, drug-loaded polymer micelles are F


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Some further studies are necessary to fine-tune therapeutic regimens for topical CsA/mPEGhexPLA treatments and to verify its suitability as a mono or combined therapy.

In the current study, the unilateral treatment with the CsA/ mPEGhexPLA nanocarrier improved EAU severity in the treated eyes of mice. This improvement correlated with proximally located immunosuppression as shown by a reduced T-cell population in cervical ipsilateral LN. In addition to these lower levels, effector functions such as proliferation and IL-2 secretion of T-cells in cervical ipsilateral LN were impaired due to the treatment. In ipsilateral LNs, elevated ConA-induced proliferation and IRBP-induced IL-2 secretion in comparison to the PBS group of ipsilateral LNs was observed but without reaching level of significance. As the inhibition of IL-2 secretion and proliferation is a central effector mechanism and is used as a pharmacodynamic measure of CsA,63−66 we used the IL-2 secretion and proliferation to measure the response to CsA in our study. Thereby, we confirmed the findings of our manuscript.35 Herein, systemic CsA treatment in EAU was associated with reduced proliferation and IL-2 secretion. Importantly, the displayed immunosuppressive effect was limited to the cervical ipsilateral LN, although contralateral and inguinal LN contained nearly similar CsA concentrations in their tissue. These findings may be related to the fact that the cervical LN received constantly the highest CsA concentration and is the effector LN to the eye. Finally, the immunosuppressive effect of CsA on splenocytes was also of minor relevance. Similar results were found after unilateral topical application of 0.5% everolimus-loaded mPEGhexPLA nanocarrier in the B10.RIII EAU model. Therein, good therapeutic efficacy of this nanocarrier in both eyes was detected as well as immunosuppressive effects in draining LNs and with lower extent also to the spleen.36 Experimental studies of drug-loaded (e.g., tacrolimus, rapamycin, infliximab, dexamethasone) delivery systems (e.g., hydrogels, liposomes, and polymeric micelles in EAU) use intravitreal injection as an application to ensure therapeutically effective drug levels.67−70 The advantage of mPEGhexPLA nanocarrier as a drug delivery system is that they can be loaded with different drugs (e.g., CsA, everolimus, glucocorticoids, and tacrolimus) and are applied noninvasively as eye drops. Due to its nontoxic characteristics, the drug-loaded mPEGhexPLA nanocarrier is suitable for the topical or systemic application and thereby is applicable for various therapeutic treatments such as ophthalmology, ulcerative colitis, and psoriasis.29,36,46,62,71 The findings of the current study supports the hypothesis that topically applied CsA/mPEGhexPLA nanocarriers target immune responses within ocular and adjacent lymphoid tissues and involve both local and systemic immunosuppressive mechanisms.

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AUTHOR INFORMATION

Corresponding Author

*Mailing Address: Maren Kasper, Department of Ophthalmology at St. Franziskus Hospital, Ophtha-Lab, Hohenzollernring 74, 48145 Münster, Germany; E-mail: [email protected]; Phone: +49-251-9352773; Fax: +49-251-9352719. ORCID

Maren Kasper: 0000-0003-2964-8866 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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ABBREVIATIONS

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REFERENCES

This work has been funded by the Ernst and Berta Grimmke Foundation, Ratingen, Germany. Projectnr. 5/13. The funding source had no involvement in study design, interpretation of data, or publication.

EAU, experimental autoimmune uveoretinitis; CsA, cyclosporine A; hIRBP, human interphotoreceptorretinoid-binding protein; p. i., postimmunization; OD, optical density; nm, nanometer; LN, lymph nodes; BSC, biopharmaceutical classification system; mPEGhexPLA, methoxy-poly(ethyleneglycol)-hexyl substituted poly(lactic acid)

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CONCLUSION Unilateral topical treatment of mice with EAU by using a CsA/ mPEGhexPLA nanocarrier formulation promoted accumulation of CsA in ocular tissues, draining, and peripheral LN, accompanied by low serum levels. Furthermore, the therapeutic CsA/mPEGhexPLA treatment suppressed the uveitogenic immune response mainly in proximal lymphatic tissues and improved EAU severity in the treated eyes. The therapeutic effect of CsA might be related to both local and systemic immunosuppression. G


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Cyclosporine A-Loaded Nanocarriers for Topical Treatment of Murine

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