Letter www.acsami.org
Ex Vivo Rat Eye Model for Investigating Transport of Next Generation Precision-Polyester Nanosystems Raghu Ganugula, Meenakshi Arora, and Majeti N. V. Ravi Kumar* Department of Pharmaceutical Sciences, College of Pharmacy, Texas A&M University, Reynolds Medical Building, TAMU Mailstop 1114, College Station, Texas 77843, United States S Supporting Information *
ABSTRACT: We present for the first time a robust ex vivo rat eye model for investigating the transport of precision-polyester nanosystems (P2Ns) across the blood−retinal barrier, intended for systemic administration. The P2Ns-GA actively transport exploiting transferrin receptors present in the inner retinal barrier and colocalize in ganglion cells. Such delivery approaches have the potential to deliver drugs to posterior segments of the eye, which is still a major challenge in treating posterior ocular disorders.
KEYWORDS: active delivery, periodic functional, polyesters, posterior segments, retinal barriers, systemic administration, transferrin receptor
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process. 20−22 Though mono or coculture models are considered valuable tools for understanding the transport properties of drugs, it is highly unlikely such models mimic tissue properties, particularly the BRB.22 Here, we present a robust ex vivo rat eyeball model for establishing the transport properties of precision-polyester nanosystems (P2Ns) intended for systemic administration. The precision polyesters (P2s) were synthesized as reported previously using a two-step polycondensation process.19 In brief, the first step consists of a synthesis of PLA-PEG prepolymer and the second step involves chain extension by incorporating an aliphatic cyclohexanetetracarboxylic dianhydride (HCDA) imparting free carboxyl groups along the polymer backbone. The P2s used in this study has a molecular weight of 30 900 (g mol−1) and 9 mol/mol carboxyl groups. These carboxyl groups were aminated and subsequently conjugated to GA following EDC chemistry.19 In addition, fluorescein coupled P2s were prepared for fluorescent studies (Figure 1a, details in SI). The fluorescein coupled P2s (4% w/ w) were mixed with P2s-GA for making fluorescent precisionpolyester nanosystems (F-P2Ns-GA) used in transport studies. The preparations rendered P2Ns-GA of ∼97 nm with GA expressed on the surface (Figure 1b,c details in SI Figure S1a,b, Figure S2). The nanosystems made using P2s (without GA) are abbreviated as P2Ns.
major challenge in drug delivery is to enhance the transport of medicines across biological barriers, such as the small intestine, the blood−retinal barrier (BRB), the blood−brain barrier, and to effectively reach the site of action.1−3 The eye is mainly divided into two segments, the anterior and posterior. Topical delivery of ophthalmic medication (eye drops) is a widely used route for treating the anterior segment of the eye, whereas its application to treat posterior conditions remain elusive.4 Effective drug delivery to the posterior segment of the eye is challenging, and alternative routes of administration (enteral, periocular, and intravitreal) are generally needed, the BRB being the major obstacle to systemic drug delivery.5−9 The intravitreal injections remain the most common method of delivering drugs to the posterior segment, whereas suprachoroidal space is being explored for low dose medications.10 Recently, research is focused on developing nanosystems for systemic administration for posterior ocular delivery, such as stealth nanosteroids, dualtargeted nanoparticles using biodegradable polymers like PLA/ PLGA.11−14 Transferrin receptors is a potential target for drug delivery to the eye, as TfR is expressed in the inner BRB, ganglion cell layer, inner nuclear layer, outer plexiform layer, photoreceptor inner segment, RPE, and choroid.15,16 Very recently, we have reported that gambogic acid (GA) coupled polyester nanosystems, fluorescent or drug encapsulated, administered orally are transported across the intestinal barriers exploiting TfR and circulate in the system facilitating distribution to TfR rich tissues.17−19 The success of novel delivery systems is highly dependent on the innovative methods of testing, early in the discovery © XXXX American Chemical Society
Received: June 2, 2017 Accepted: July 24, 2017 Published: July 24, 2017 A
DOI: 10.1021/acsami.7b07896 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
ACS Applied Materials & Interfaces
Figure 1. (a) Hypothetical reaction scheme for GA/fluorophore conjugation to P2s. (b) DLS particle size distribution of P2Ns [scale and text are added manually superimposing the original figure for better clarity]. (c) UV/vis spectra of P2Ns-GA and GA shows GA characteristic peaks at 290 and 360 nm. However, such peak was not present in P2s (without GA).
To demonstrate the transport of P2Ns, we have used rat ex vivo eyeball model that represents both anterior and posterior segments and the respective barriers intact (Figure S3a−d). Our results indicate that the P2Ns-GA made their way to retinal space and localized in ganglion cell region, while P2Ns (without GA) did not (Figure 2a,b). The P2Ns-GA appears to bind to the erythrocytes in the inner retinal microvasculature and cross the inner retinal barrier (Figure 2c, Figure S4, Figure S5 [Video S1]). We can also see some P2Ns and P2Ns-GA in the capillaries in the outer plexiform layer (Figure 2a). We have also observed the presence of both P2Ns and P2Ns-GA in the retinal pigment epithelium, the outer retinal barrier (Figure S6). The P2Ns and P2Ns-GA are also seen in the choroidal space (Figure S6). A significant number of P2Ns-GA presence was observed in the ciliary process that generates aqueous humor (Figure S7). Interestingly, the majority of P2Ns and not P2Ns-GA, adhered to the cornea (Figure S8a,b). To understand the mechanisms of the P2Ns-GA transport, we have labeled transferrin receptor, specific to GA in the tissue sections. The P2Ns-GA colocalize with erythrocytes in the inner retinal vasculature leading to ganglion cells (Figure 3, Figures S9, 10, S11 [Video S2]). The P2Ns-GA also colocalize in the RPEs expressing TfR, whereas we do not see any in the choroidal region (Figure S12). The TfR staining was evident in the stromal region of the cornea; however, we have not observed P2Ns-GA transport (Figure S8c,d). The presence of P2Ns-GA in the retinal ganglion cells was further confirmed by labeling Glial fibrillary acidic protein (GFAP) (Figure 4a,b). The P2Ns-GA are again seen associated with erythrocytes in the retinal microvasculature (Figure 4c,d, Figure S13, Figure S14 [Video S3]). The P2s, unlike conventional terminal polyesters, offered periodic functionality along the polymer backbone that in turn facilitated enhanced ligand coupling.19 The P2s-GA upon emulsification with 4% fluorescent P2s led to precision polymer nanosystems (P2Ns-GA) of 100−125 nm in size with surface
Figure 2. Retinal sections from the eyeballs exposed to different treatments. (a) Retina was immunostained with actin (red) and nucleus was stained with DAPI (blue). (b) Array of ganglionic cell layer (GCL) (scale bar: 2 μm). (c) Sequential images (F-P2Ns-GA 1− 16) generated through z-stacking (0.9 μm thickness distance for each) showing vasculature (inner blood retinal barrier (IBRB) and GCL (scale bar: 10 μm).
Figure 3. Transferrin receptor colocalization of F-P2Ns in the retina. (a) F-P2Ns-GA and not F-P2Ns show colocalization with TfR (red), nucleus DAPI (blue); (b) higher magnification image of the highlighted area within the GCL space for control and P2Ns-GA, whereas the highlighted area in the P2Ns group is toward RPE (scale bar: 2 μm).
GA that was confirmed by GA characteristic peaks in the UV− vis region at 290 and 360 nm. The use of intact fresh eyeballs (providing three-dimensional access to the P2Ns) allowed us to better understand the ability of P2Ns-GA transport across the BRB. The P2Ns (no GA) B
DOI: 10.1021/acsami.7b07896 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
ACS Applied Materials & Interfaces
permeation across the epithelium, though partial,24 and our results are in agreement with residence time but not permeation. The P2Ns seen in the RPE, outer blood−retinal barrier did not appear to have permeated to the retina. However, achieving RPE delivery by systemic means is also a significant challenge that the P2Ns can overcome. The P2Ns to RPE appears to have taken choroidal vasculature. The presence of P2Ns-GA in the ciliary process suggests the involvement of long ciliary arteries in the transport. The P2Ns-GA exploited the surface GA, which has the full potential to facilitate TfR-mediated transport. Recently, we have shown P2Ns-GA or PLGA-GA nanosystems facilitate noncompetitive transport across the intestinal barriers exploiting TfR leading to enhanced bioavailability and modular tissue distribution.17−19 Here we have seen that the P2Ns-GA are associated with erythrocytes though the nature of the association is not clear from the actin staining sections. However, the appearance of the P2Ns-GA in the ganglion cell region confirms the release of P2Ns-GA from the erythrocytes and possibly involving TfR-mediated transport across the endothelium. It is interesting to note that the P2Ns-GA shows significant colocalization within erythrocytes in sections stained for TfR, though literature reports suggest that matured erythrocytes do not express TfR.25 Similarly, we have seen the GFAP also stained erythrocytes and the P2Ns-GA colocalize within the erythrocytes. The literature evidence suggests that binding to erythrocytes prolong the circulation times of the particles and it is important to have these particles bind reversibly to the erythrocytes if they have to be targeted to cell or tissue of interest.26,27 All these data sets establish the ability of P2Ns-GA crossing the inner retinal barriers making their way ganglion cells. Though we could not establish from the available data sets, if P2Ns-GA have crossed the RPEs to enter retina, we have certainly established their ability to cross the inner retinal barriers. In summary, our data represents unique P2Ns-GA that can facilitate TfR mediated transport across BRB, and in doing so, there is tremendous opportunity in realizing systemic delivery to posterior segments of the eye.
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ASSOCIATED CONTENT
S Supporting Information *
Figure 4. Glial fibrillary acidotic protein (GFAP) colocalization of FP2Ns-GA in the retina. (a) F-P2Ns-GA and not F-PN2s shows colocalization with GFAP (red), nucleus DAPI (blue); (b) higher magnification image of the highlighted area within the GCL space; (c) sequential images retina (1−12). The images were generated through z-stacking (0.46 μm thickness distance for each). The P2Ns-GA colocalize with GFAP in ganglion cells. GFAP immunofluorescence in ganglion cells and the RBCs in blood vessel in the puncta of images emphasize the transport within the IBRB (scale bar: 5 μm); (d) FP2Ns-GA colocalize within RBCs and the zoomed areas showing FP2Ns-GA cross the IBRB making their way to inner nuclear layer as well as GCL (scale bars: 2 μm).
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b07896. Experimental and characterization data (PDF) Video S1: Actin section slices (AVI) Video S2: TfR section slices (AVI) Video S3: GFAP section slices (AVI)
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AUTHOR INFORMATION
Corresponding Author
*M. N. V. R. Kumar. E-mail:
[email protected]; Tel.: (979) 436-0721.
were seen adhering to the corneal layer due to hydrogenbonding of acidic carboxyl groups of P2Ns with corneal surface and do not seem to have permeated the corneal epithelium. The literature reports23 support these findings, in which transferrin-coupled polystyrene nanoparticles are seen highest in the corneal epithelium and in the study conducted without corneal epithelium resulted in significant uptake in the corneal stroma. There also are reports suggesting surface charge, mostly cationic, of the nanoparticles improve corneal residence or
ORCID
Raghu Ganugula: 0000-0003-0262-5170 Majeti N. V. Ravi Kumar: 0000-0001-5606-401X Funding
Authors acknowledge, the College of Pharmacy, TAMU for a seed grant. Notes
The authors declare no competing financial interest. C
DOI: 10.1021/acsami.7b07896 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
ACS Applied Materials & Interfaces
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ACKNOWLEDGMENTS We acknowledge Dr. Ficht, HSC for providing access to particle size analyser; Dr. Mouneimne, Image analysis laboratory for the help with confocal image analysis.
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ABBREVIATIONS DLS, dynamic light scattering EDA, ethylenediamine EDC, 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide F-NS, fluorescent nanosystems GA, gambogic acid NMR, nuclear magnetic resonance P2s-GA, GA-functionalized P2s P2Ns, precision-polyester nanosystems P2Ns-GA, precision-polyester nanosystems-GA transferrin, and transferrin receptor
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DOI: 10.1021/acsami.7b07896 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
