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Nanoparticle–integrin antagonist C16Y peptide treatment of choroidal neovascularization in rats
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Journal of Controlled Release 142 (2010) 286–293
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Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l
Nanoparticle–integrin antagonist C16Y peptide treatment of choroidal neovascularization in rats Hyuncheol Kim a,⁎, Karl G. Csaky b a b
Chemical and Biomolecular Engineering, Sogang University, #1 Shinsu-dong Mapo-gu, Seoul 121-742, Republic of Korea Department of Ophthalmology, Duke University Medical Center, Durham, North Carolina, USA
a r t i c l e
i n f o
Article history: Received 28 July 2009 Accepted 29 October 2009 Available online 4 November 2009 Keywords: Choroidal neovascularization Nanoparticles Integrin antagonist Intravitreal injection
a b s t r a c t Choroidal neovascularization (CNV) is the major cause of severe vision loss in patients with age-related macular degeneration (AMD). Present drug delivery may be limited by poor delivery to the choroid where CNV originates. The goal of this study was to develop a drug delivery system to deliver an integrin– antagonist peptide to the sub-retinal space. We developed polylactic acid/polylactic acid–polyethylene oxide nanoparticles (PLA/PLA-PEO) encapsulating the water-soluble integrin–antagonist peptide, C16Y (C16Y-NP). The PLA/PLA-PEO nanoparticles were 302 +/− 85.1 nm in size and demonstrated a two-week sustained release, in vitro, of encapsulated C16Y. Injected nanoparticles did not demonstrate retinal toxicity as determined by histopathology. C16Y peptide solution or C16Y-NP was injected 5 or 9 days post laser photocoagulation. A single intravitreal injection of C16Y peptide and C16Y-NP solution at both 5 days and 9 days post laser photocoagulation statistically inhibited CNV (p b 0.05). However, for the day 5 injections the area of choroidal neovascularization on day 12 was smaller for C16Y-NP than for C16Y peptide solution (p b 0.05) because of the short vitreous half-life of C16Y peptide solution. These results demonstrate the importance of sustained release delivery for the treatment of choroidal neovascularization associated with age-related macular degeneration. The intravitreally administered PLA/PLA-PEO containing coumarin was found to penetrate the retina and localize to the RPE. These results suggest that nanoparticles of biodegradable polymers may be a potential useful delivery system for intravitreal injection of drugs in the treatment of AMD. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Choroidal neovascularization (CNV) is the major cause of irreversible vision loss associated with age-related macular degeneration (AMD) [1]. Several therapies such as laser photocoagulation, photodynamic therapy, macular translocation and surgical removal are available to treat AMD [2]. However, these therapies usually fail because of CNV recurrence. Therefore, pharmacologic therapy has been developed to inhibit CNV. However, as CNV originates from the choroid, delivering therapeutic agents to the choroid and the retinal pigment epithelium to inhibit CNV is challenging because of several formidable barriers. Barriers for topical drug administration include tear film washout, physical properties of the cornea, the long diffusion distance between the cornea and retina and the rapid clearance by aqueous humor flow [3,4]. The transscleral drug delivery route is also limited by several active episcleral clearance mechanisms [5,6]. Indeed, placement of the anti-angiogenic agent anecortave acetate onto the sclera for the treatment of CNV was shown not to be effective
⁎ Corresponding author. Tel.: +82 2 705 8922; fax: +82 2 3273 0331. E-mail address: [email protected] (H. Kim). 0168-3659/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2009.10.031
[7]. Directly injecting drugs into the vitreous has shown favorable results, in multiple clinical trials, in delivering therapeutic agents to the posterior segment of the eye [8–10]. However, intravitreal injections invariably need to be repeated on a frequent basis and this approach is associated with potential serious complications including vitreous hemorrhage, retinal detachment, and endophthalmitis [11,12]. Therefore, sustained release intravitreal injectable delivery methods such as injected bioerodible or nonbioerodible systems have been developed [13,14] but do not address the issue of retinal penetration. The integrin heterodimers, αvβ3, αvβ5, and α5β1, play important roles in the pathogenesis of angiogenesis [15,16]. CNV predominantly expresses integrin αvβ3 and blocking this integrin heterodimer has been reported to inhibit neovascularization [17,18]. The scrambled C16Y peptide has been previously shown to block both the αvβ3 and α5β1 integrins and reduce chick chorioallantoic membrane angiogenesis and tumor growth [19]. Because peptides typically have very short intravitreal half-lives, in the present study we have developed biodegradable polymer-based nanoparticles to deliver the angiogenesis inhibitor peptide, C16Y, to the posterior segment of the eye. The aim of the study was to obtain a more sustained delivery of the peptide and improve retinal penetration of the drug.
2. Materials and methods 2.1. Materials Poly(D,L-lactide) (PLA) was purchased from Birmingham Polymers (Birmingham, AL, USA). Poly(ethylene oxide-b-lactide) (DL form) (PEO(700)-b-PLA(3750)) was purchased from Polymer Source (Polymer Source Inc., Dorval, Quebec). C16Y peptide (DFKLFAVYIKYR) was synthesized by Celtek Peptide (Celtek Bioscience, LLC., Nashville, TN, USA). Polyvinyl alcohol (PVA, 88% mol hydrolyzed, MW 31,000) was obtained from Fluka (Sigma-Aldrich Corp., St. Louis, MO, USA). All other chemicals were of reagent grade. 2.2. Nanoparticle formulation The nanoparticle preparation method was a modification of the W1/O/W2 emulsion method previously described [20]. Briefly, 5.34 mg of C16Y peptide was dissolved in 0.3 mL of formamide. 50.0 mg of PLA was dissolved in 2 mL of methylene chloride. 50 µL of PLA-PEO acetone solution (0.1 g/mL) was added to the PLA organic solution. 0.3 mL of C16Y peptide solution (17.8 mg/mL) was added and emulsified in the PLA and PLA-PEO organic solution by sonicating with 20 W for 30 s. 10 mL of 5% (w/v) PVA solution was added to the first emulsion and then the solution was sonicated with 20 W for 30 s. This emulsion was gently poured into 50 mL of a 0.5% (w/v) PVA solution with magnetic bar stirring. Then, the emulsion was stirred overnight to evaporate the organic solvents. Nanoparticles were recovered by centrifugation at 16,000 RPM for 40 min and washed with ultrapure water. This process was repeated three times to remove residual PVA and organic solvents and the nanoparticles were freeze-dried. To determine the distribution of intravitreally administered PLA/ PLA-PEO nanoparticles in the retina, a lipophilic fluorescent marker, 6-coumarin, was incorporated in the nanoparticles. The dye was reported to associate with the polymer matrix of nanoparticles and not leach from the nanoparticles [21]. The fluorescence dye, coumarin (0.2 mg) was dissolved in 50.0 mg of PLA organic solution and coumarin encapsulating PLA/PLA-PEO nanoparticles were fabricated as described above. 2.3. Particle size analysis Particle size was determined by photon correlation spectroscopy (PCS) using quasi-elastic light scattering equipment (ZetaPlus™ equipped with particle sizing mode, Brookhaven Instrument Corp., Holtsville, NY, USA). A dilute suspension of nanoparticles was prepared in ultrapure water. The sample was subjected to particle size analysis and analyzed in duplicate with 10 readings per nanoparticle sample. Zeta potential of nanoparticles in distilled water was determined using ZetaPlus™ in the zeta potential analysis mode in duplicate with 10 readings per nanoparticle sample. Morphological evaluation of the nanoparticles was performed using scanning electron microscopy (SEM) (Hitachi S-4500). 2.4. In vitro release study In vitro release of C16Y from nanoparticles was determined as previously described [22]. 18.0 mg of nanoparticles (containing 1.07 mg of C16Y peptide) were placed in a conical tube containing 1.5 mL of ultrapure water and incubated at 37 °C under continuous shaking. At various time intervals, the sample was centrifuged at 16,000 RPM for 40 min and 0.7 mL of the supernatant was analyzed by HPLC. The peptide release study was continued after replacement with the same volume of fresh ultrapure water and resuspension by vortexing. In vitro release rates from the nanoparticles were determined by assaying the peptide concentrations over time with a
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reverse-phase high-performance liquid chromatography (HPLC) assay. Samples or standards with volumes of 5 to 200 µL were injected with an autosampler (model G1329A; Agilent Technologies, Palo Alto, CA, USA). A 250 × 4.6 mm (5 µm) C18 polymeric column (Vydac, Hesperia, CA, USA) was used for separation. Separation was conducted by 1.0 mL/min isocratic elution of water with 0.1% TFA and acetonitrile with 0.1% TFA by volume and a pump (model G1312A; Agilent). The concentrations of the samples were monitored at 230 nm with a UV detector (model G115A; Agilent) and analyzed on computer (Chemstation software; Agilent). The retention time was 8.1 min. The standard curve was linear (R2 = 0.99) over the range of 100 to 75,000 ng/mL. The drug detection limit for the peptide in solvent was 10 ng/mL. Peptide release profiles were generated for each nanoparticle formulation in terms of cumulative peptide release versus time. 2.5. Argon laser-induced CNV in rats All animal studies conformed to the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. Male brown Norway rats (N = 18) aging 8–12 weeks were anesthetized by intramuscular injection of ketamine (70 mg/kg) and xylazine (30 mg/kg) and both pupils were dilated with 1% Tropicamide (Mydriacyl®, Alcon, Fort Worth, TX, USA). Hydroxypropyl methycellulose (DECA Pharmaceuticals, LLC., Bowling Green, KY, USA) was applied to each eye and a microscope cover glass was used as a contact lens. Both eyes received four laser burns between retinal vessels around the optic nerve head using the blue-green setting of a Lumenis Ultima Argon Laser (Lumenis Inc., New York, NY, USA) using a laser power of 180 mW for 0.1 s and a spot size of 50 μm. 2.6. Effect of intravitreal C16Y encapsulating nanoparticle on experimental CNV Laser-induced CNV has been demonstrated to grow in size reaching a maximum area at 2 weeks after laser treatment. On day 0, laser photocoagulation was performed and either on day 5 (n = 6—each group) or day 9 (n = 3—each group), 10 μL of PBS alone or PBS containing empty nanoparticles (0.12 mg/μL), C16Y (3 μg/μL), or C16YNP (0.12 mg/μL) was injected. The materials were injected intravitreally with a 32-gauge needle after topical anesthesia with 1% tropicamide ophthalmic solution (Mydriacyl®, Alcon, Forth Worth, Texas, USA). On day 12, the animals were euthanized by CO2 inhalation. 2.7. Visualization and quantification of CNV Blood vessels were visualized using fluorescent isothiocyanate (FITC)-dextran (MW = 2 × 106 Da) perfusion and preparation of choroidal flat mounts as described previously [23,24]. Briefly, the eyes were sectioned at the equator and the anterior segment of the eye, vitreous and retina were removed. The posterior segment of the eye including the sclera and the choroid, was dissected into quarters by four radial cuts and mounted on a microscope slide with a fluorescent mounting media (Vectashield®, Vector Laboratories, Inc., Burlingame, CA, USA). Images of the flat mounts were captured with a fluorescent microscope (DM5000B, Leica Microsystems Inc, Bannockburn, IL, USA) using a FITC filter (excitation 471 nm and emission 503 nm) and a CCD camera coupled to a computer with image analysis software (Volocity 3.6.1, ImproVision, Inc., Lexington, MA). Blood vessels within the neovascular lesion that were labeled with FITC-dextran were highlighted and converted into a binary image with ImageJ 1.34S (National Institutes of Health, Bethesda, MD, USA). The area occupied by black pixels was calculated with Matlab software (version 6.5.1, The MathWorks, Inc., Natick, MA, USA).
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2.8. Determination of the retinal distribution of intravitreally administered PLA/PLA-PEO nanoparticles 10 μL of a suspension of PLA/PLA-PEO nanoparticles encapsulating coumarin dye (18.5 μg/μL) was injected into the vitreous as described above. The eyes were enucleated and frozen in the freezing mold with optimal cutting temperature (O.C.T) compound 6 h, 1 day, and 3 days post intravitreal injection. Then, the eyes were kept at − 20 °C overnight and 10 μm sections were generated. The distribution of nanoparticles in the retina was examined under an epifluorescence microscope (BX50; Olympus, Tokyo, Japan). 2.9. Toxicity of nanoparticle Nanoparticles were manufactured as described above. 10 μl of nanoparticles (0.12 mg/μL) were injected in the right eyes of three rats. Three days post injection, the eyes were enucleated and frozen immediately using the OCT compound. The frozen eyes were sectioned with a cryotome and then fixed with 4% PFA in 1× PBS buffer. The sectioned eyes were stained with hematoxylin and eosin for light microscopic examination. 2.10. Statistical analysis The differences in the mean area of choroidal neovascularization at each group from the control group were compared and tested by the analysis of variance (ANOVA) using PSI-Plot version 7.3 (Poly software International, Inc., Pearl River, NY, USA). Significant differences were determined for p b 0.05. 3. Results 3.1. Nanoparticle analysis and toxicity PLA/PLA-PEO nanoparticles containing C16Y peptide demonstrated a bimodal size distribution in two size ranges: 114–126 nm and 302–367 nm (Fig. 1A). The average zeta potential was measured to be −38.26 +/− 1.42 mV (mean +/− SE, n = 10) in ultrapure water (Fig. 1B). Fig. 1C demonstrates a consistent spherical shape of generated PLA/PLA-PEO nanoparticles as seen by scanning electron microscopy. The measured amount of non-encapsulated C16Y peptide in the supernatant during the purification was found to be 2.37 mg. The starting amount of C16Y peptide was 5.34 mg, giving an encapsulation efficiency of 55.6%. The release of C16Y from the PLA/PLA-PEO nanoparticles in pure water was measured in vitro at 37 °C. 19 mg of nanoparticles containing 1.07 mg of C16Y were placed into pure water. The nanoparticles exhibited sustained release of the encapsulated C16Y peptide over 6-weeks (Fig. 2). To test for retinal toxicity, retinal histology was performed following intravitreal injection of PLA/PLA-PEO. The treated eyes (Fig. 3A) showed no evidence of inflammatory cells or loss of normal retinal architecture, compared to the control eye (Fig. 3B). 3.2. Angiographic analysis of RPE–choroid–sclera flat mount Both C16Y peptide solution and C16Y-NP were evaluated with a rat laser model of CNV. High molecular weight FITC-dextran was perfused to visualize the choroidal neovascularization. Figs. 4 and 5 show the representative laser-induced CNV from each group on day 12 post laser photocoagulation following intravitreal injection on day 5 or day 9, respectively. There was no difference in the neovascular area between the PBS alone and PBS containing nanoparticle groups injected on day 5 (p N 0.05) and day 9 (p N 0.05) post laser photocoagulation, respectively
Fig. 1. Particle analysis and electron microscope photograph. The size and surface charge of C16Y encapsulating PLA/PLA-PEO nanoparticles were 302.5 ± 85.1 nm (A) and − 38.26 ± 1.42 mV (B), respectively. (C) The nanoparticles containing C16Y peptide exhibited a spherical shape.
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3.3. Retinal distribution of intravitreally administered PLA/PLA-PEO nanoparticles PLA/PLA-PEO nanoparticles encapsulating the fluorescence dye (coumarin) diffused into the outer plexiform layer by 6 h post intravitreal injection (Fig. 6A) and were endocytosed into the RPE by 24 h (Fig. 6B). Nanoparticles were still retained in the retina 3 days after intravitreal administration (Fig. 6C). The results demonstrated that PLA/PLA-PEO nanoparticles over 100-nm in size easily penetrate the retina structure into the RPE following intravitreal injection. 4. Discussion
Fig. 2. PLA/PLA-PEO nanoparticles release C16Y peptide over six weeks in vitro.
(Figs. 4E and 5E). Empty PLA/PLA-PEO nanoparticles injected intravitreally at either day 5 or day 9 was found to have no antiangiogenic activity. A single intravitreal injection of C16Y peptide and C16Y-NP solution at both 5 days and 9 days post laser photocoagulation statistically inhibited CNV (p b 0.05). However, C16Y-NP solution injected on day 5 reduced the area of choroidal neovascularization more than the C16Y peptide solution injected on day 5 (p b 0.05).
Fig. 3. Representative histopathologic sections through the retina region of (A) the rat eye three days post intravitreal injection of PLA/PLA-PEO nanoparticles and (B) the non-treated rat eye. No inflammatory response or toxicity to the photoreceptors was observed (stain, hematoxylin and eosin). GCL, IPL, INL, OPL, ONL, PRL, and RPE represent the ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, photoreceptor layer, and retinal pigment epithelium, respectively.
Intravitreally administered micro- or nano-sized particles were reported to be able to deliver therapeutic agents to the posterior segment of the eye for an extended period of time [25–27]. In this study, we evaluated the effect of intravitreal injection of PLA/PLA-PEO nanoparticles encapsulating the anti-angiogenic C16Y peptide on inhibition of experimental CNV. The intravitreal C16Y peptide could be expected to diffuse easily into the sub-retinal space following intravitreal injection because the peptide's molecular weight (MW = 1600 Da) is much smaller than the maximum size (around 80 kDa) of solutes, that are readily able to penetrate the retina structure [28,29]. Both the C16Y peptide and C16Y-NP solutions, which were injected intravitreally on days 5 and 9 post laser photocoagulation, inhibited the experimental CNV, compared to the control (p b 0.05). However, C16Y-NP injected on day 5 was found to inhibit the choroidal neovascularization dramatically more than the C16Y peptide solution on day 5 (p b 0.05). In the C16Y peptide solution, 30 µL of C16Y peptide was injected. On the other hand, 1.2 mg of C16Y-NP could be expected to deliver 1.16 µg of C16Y peptide for 7 days, according to the in vitro release study. Although the C16Y solution injection delivered 25-fold more peptide than that estimated for C16Y-NP injection, the choroidal neovascularization was inhibited less with the C16Y peptide solution (p b 0.05) (Fig. 4E). The elimination of intravitreally administered water-soluble C16Y peptide was so active that the half-life of intravitreal C16Y peptide solution was not long enough for the peptide injected on day 5 post laser photocoagulation to inhibit the experimental CNV as much as the C16Y-NP injected on day 5 did. This indicates the importance of sustained release delivery for the treatment of choroidal neovascularization associated with age-related macular degeneration. The elimination route from the vitreous can be by diffusion into the anterior and then drainage through the aqueous pathway or across the retina. The half-life of intravitreal C16Y can be estimated to be around 10 h from half-lives of other agents [30]. Most of the C16Y peptide administered intravitreally in solution might then be eliminated within two days, which could account for the lesser inhibition of experimental CNV than that observed following C16Y-NP administration. C16Y-NP delivered the therapeutic agent long enough to inhibit the experimental CNV following intravitreal injection on both day 5 and day 9 post laser photocoagulation (p b 0.05). These results demonstrate that biodegradable polymer-based nanoparticles may be a potential intravitreal drug delivery system for the treatment of choroidal neovascularization associated with age-related macular degeneration. However, the optimal release kinetics of the biodegradable polymer-based nanoparticles should be investigated with a modified polymer ratio before clinical application. Polylactic acid polymer has been investigated for ocular drug delivery because of biocompatibility [31,32]. Intravitreally administered PLA microparticles were non-toxic at ocular tissues [27,33]. On the other hand, intravitreally injected PLA nanoparticles induced mild inflammation at the retina at early time points [26,34]. PLA polymer is known to induce mild and chronic inflammatory responses in soft tissues [35,36]. Intravitreal microparticles remain suspended in the vitreous. Intravitreal nanoparticles, on the other hand, diffuse to the
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Fig. 4. Representative images of anterior segments dissected on day 12 post laser photocoagulation. Each image is centered about a 50 µm diameter laser spot region. Each group of animals received intravitreal injection on day 5 of: (A) PBS alone; (B) PBS containing empty PLA/PLA-PEO nanoparticles; (C) C16Y peptide solution; (D) C16Y peptide encapsulating PLA/PLA-PEO nanoparticles. (E) Image analysis demonstrates that a single intravitreal injection of C16Y peptide and C16Y-NP solution at both 5 days and 9 days post laser photocoagulation statistically inhibited CNV (p b 0.05). However, for injection on day 5, the area of choroidal neovascularization on day 12 was smaller for C16Y-NP than for C16Y solution (p b 0.05).
retina and accumulate on the inner limiting membrane with some particles diffusing deeper into the retina [37]. So, we hypothesized that the inflammation response of the PLA based nanoparticles came from direct interaction between hydrophobic PLA polymer and ocular tissues. Inflammation response has been known to negatively influence retina physiology [38]. Hydrogels, such as polyethylene
glycol (PEG) and polyethylene oxide (PEO), have been known to induce no inflammation response at soft tissues [39]. So, we blended polylactic acid with a co-polymer of PLA (MW = 700) and PEO (MW = 3750) to reduce the contact of naked PLA and retina tissue. PLA, the hydrophobic part of the co-polymer, is embedded into the organic phase; on the other hand, PEO, the hydrophilic part of the co-
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Fig. 5. Representative images of anterior segments dissected on day 12 post laser photocoagulation. Each image is centered about a 50 µm diameter laser spot region. Each group of animals received intravitreal injection on day 9 of: (A) PBS alone; (B) PBS containing empty PLA/PLA-PEO nanoparticles; (C) C16Y peptide solution; (D) C16Y peptide encapsulating PLA/PLA-PEO nanoparticles. (E) Image analysis demonstrates that both intravitreally injected C16Y-NP and C16Y peptide solution injected on day 9 inhibited the CNV significantly (p b 0.05) compared to the control.
polymer, is directed into the aqueous phase around the PLA organic droplets during the nanoparticle fabrication [40]. The toxicity study showed no inflammatory response from PLA/PLA-PEO nanoparticles in the retinal tissue on day 3 post intravitreal injection (Fig. 3). In the current study, we could reduce the toxicity of a PLA nanoparticle by decreasing the direct contact of PLA and retinal tissue with PEO. However, the intravitreally administered PLA/PLA-PEO nanoparticles degrade with time to increase the contact of PLA and retinal tissue.
The increased contact might induce an inflammatory response in the ocular tissues at a later time than for the observations in the present study. The evaluation of long-term toxicity should be performed before clinical trials and remains for future work. The αvβ3 integrin is known to play an important role in angiogenesis and to express predominantly in neovascular ocular tissues in age-related macular degeneration patients [15,16,41–43]. Blocking the ligation of integrin into the extracellular matrix has been
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Recently, nano-sized drug delivery carriers have been intensively investigated for delivering therapeutic agents to the posterior segment of the eye [13]. However, the information on the distribution of intravitreally administered nanoparticles is limited. Upon intravitreal administration, many nanoparticles first settled down on the inner limiting membrane (ILM), which might be the major barrier to the penetration of intravitreal nanoparticles into the retina [34]. And then, some nanoparticles passed through the ILM penetrating deeper retina structures and were observed in the RPE for several months [26,34,47]. Some retina layers show formidable barriers against the trans-retinal penetration of macromolecules. For example, the ILM is composed of a fine three-dimensional meshwork structure with numerous pores whose size ranges from 10 to 25 nm [48]. And the external limiting membrane (ELM) is formed by rows of zonulae adherents, whose pore radius is between 30 and 36 Å [49]. However, this study demonstrated that intravitreally administered PLA/PLAPEO nanoparticles greater than 100 nm in diameter penetrate the retinal structures (Fig. 6). Our previous study demonstrated that the Müller cells might play an important role in the retinal penetration of human serum albumin nanoparticles [47]. So, we surmise that PLA/ PLA-PEO nanoparticles penetrate through the Müller cells. However, the verification of the trans-retinal penetration mechanism of the PLA/PLA-PEO nanoparticles was beyond the scope of this study. Sakurai et al. [37] reported that intravitreally administered polystyrene nanoparticles were distributed homogeneously and the half-lives of intravitreal 2 μm, 200 nm, or 50 nm polystyrene were 5.4, 8.6, and 10.1 days, respectively. We found in the current study that the intravitreally administered PLA/PLA-PEO nanoparticles dispersed in the vitreous and thus might cause vision discomfort to patients. In addition, many of the nanoparticles were found in the ciliary body and the trabecular meshwork. The nanoparticles are able to enter cells easily by endocytosis and pinocytosis and then are degraded into biocompatible monomer [50]. Nanoparticles inside the cells may induce disturbance of the aqueous humor circulations. Although the PLA/PLA-PEO nanoparticles did not provoke inflammation, the interaction between endocytosed nanoparticles and ocular cells must be investigated intensively before clinical application. 5. Conclusions In conclusion, we developed non-toxic ocular nanoparticles by blending PLA and PLA-PEO co-polymer together to deliver the watersoluble integrin–antagonist peptide, C16Y, to the sub-retinal space in order to inhibit the experimental choroidal neovascularization. The nanoparticle based C16Y peptide delivery was superior to C16Y peptide solution delivery. Fig. 6. The retinal distribution of intravitreally administered coumarin encapsulating PLA/PLA-PEO nanoparticles (A) 6 h; (B) 1 day; (C) 3 days post injection. (B) Inset demonstrates the endocytosis of the nanoparticles in the RPE. GCL, IPL, INL, OPL, ONL, PRL, and RPE represent the ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, photoreceptor layer, and retinal pigment epithelium, respectively. All images were captured at × 20 magnification (inset at × 60).
proven to prevent blood vessel formation in several in vivo models [15,44]. RGD motif peptides target and block αvβ3 [2,45,46]. The intravitreal injection of 200 μg or 100 μg RGD peptide 9 days and 11 days post laser photocoagulation inhibited choroidal neovascularization efficiently. However, the intravitreal injection of 50 μg RGD peptide did not inhibit the CNV, compared to the 0 μg control injection. On the other hand, the intravitreal injection of 30 μg of C16Y peptide on 9 days post laser photocoagulation showed statistically significant difference from the PBS alone injection control group. Therefore, the C16Y peptide appears to be more efficient at inhibiting CNV than RGD based peptide.
Acknowledgements The work is supported in part by Sogang University internal research grant (200910005.01). The authors are grateful to Dr. Peter M. Bungay, National Institutes of Health, for his kind help and useful suggestions. References [1] F.L. Ferris 3rd, S.L. Fine, L. Hyman, Age-related macular degeneration and blindness due to neovascular maculopathy, Arch. Ophthalmol. 102 (1984) 1640–1642. [2] S.L. Fine, J.W. Berger, M.G. Maguire, A.C. Ho, Age-related macular degeneration, N. Engl. J. Med. 342 (2000) 483–492. [3] C.L. Bourlais, L. Acar, H. Zia, P.A. Sado, T. Needham, R. Leverge, Ophthalmic drug delivery systems—recent advances, Prog. Retin. Eye Res. 17 (1998) 33–58. [4] A.A. Moshfeghi, G.A. Peyman, Micro- and nanoparticulates, Adv. Drug Deliv. Rev. 57 (2005) 2047–2052. [5] H. Kim, M.R. Robinson, M.J. Lizak, G. Tansey, R.J. Lutz, P. Yuan, et al., Controlled drug release from an ocular implant: an evaluation using dynamic three-dimensional magnetic resonance imaging, Invest. Ophthalmol. Vis. Sci. 45 (2004) 2722–2731.
[6] M.R. Robinson, S.S. Lee, H. Kim, S. Kim, R.J. Lutz, C. Galban, et al., A rabbit model for assessing the ocular barriers to the transscleral delivery of triamcinolone acetonide, Exp. Eye Res. 82 (2006) 479–487. [7] J.S. Slakter, T.W. Bochow, D.J. D'Amico, B. Marks, J. Jerdan, E.K. Sullivan, et al., Anecortave acetate (15 milligrams) versus photodynamic therapy for treatment of subfoveal neovascularization in age-related macular degeneration, Ophthalmology 113 (2006) 3–13. [8] D.M. Brown, P.K. Kaiser, M. Michels, G. Soubrane, J.S. Heier, R.Y. Kim, et al., Ranibizumab versus verteporfin for neovascular age-related macular degeneration, N. Engl. J. Med. 355 (2006) 1432–1444. [9] E.S. Gragoudas, A.P. Adamis, E.T. Cunningham Jr, M. Feinsod, D.R. Guyer, VEGF inhibition study in ocular neovascularization clinical trial group. , Pegaptanib for neovascular age-related macular degeneration, N. Engl. J. Med. 351 (2004) 2805–2816. [10] P.J. Rosenfeld, D.M. Brown, J.S. Heier, D.S. Boyer, P.K. Kaiser, C.Y. Chung, et al., Ranibizumab for neovascular age-related macular degeneration, N. Engl. J. Med. 355 (2006) 1419–1431. [11] J.I. Lim, R.A. Wolitz, A.H. Dowling, H.R. Bloom, A.R. Irvine, D.M. Schwartz, Visual and anatomic outcomes associated with posterior segment complications after ganciclovir implant procedures in patients with AIDS and cytomegalovirus retinitis, Am. J. Ophthalmol. 127 (1999) 288–293. [12] T.S. Shane, D.F. Martin, Endophthalmitis after ganciclovir implant in patients with AIDS and cytomegalovirus retinitis, Am. J. Ophthalmol. 136 (2003) 649–654. [13] R.C. Nagarwal, S. Kant, P.N. Singh, P. Maiti, J.K. Pandit, Polymeric nanoparticulate system: a potential approach for ocular drug delivery, J. Control. Release 136 (2009) 2–13. [14] K. Hironaka, Y. Inokuchi, Y. Tozuka, M. Shimazawa, H. Hara, H. Takeuchi, Design and evaluation of a liposomal delivery system targeting the posterior segment of the eye, J. Control. Release 136 (2009) 247–253. [15] P.C. Brooks, R.A. Clark, D.A. Cheresh, Requirement of vascular integrin alpha v beta 3 for angiogenesis, Science 264 (1994) 569–571. [16] M. Friedlander, C.L. Theesfeld, M. Sugita, M. Fruttiger, M.A. Thomas, S. Chang, et al., Involvement of integrins alpha v beta 3 and alpha v beta 5 in ocular neovascular diseases, Proc. Natl Acad. Sci. USA 93 (1996) 9764–9769. [17] H. Kamizuru, H. Kimura, T. Yasukawa, Y. Tabata, Y. Honda, Y. Ogura, Monoclonal antibody-mediated drug targeting to choroidal neovascularization in the rat, Invest. Ophthalmol. Vis. Sci. 42 (2001) 2664–2672. [18] N. Umeda, S. Kachi, H. Akiyama, G. Zahn, D. Vossmeyer, R. Stragies, et al., Suppression and regression of choroidal neovascularization by systemic administration of an {alpha}5{beta}1 integrin antagonist, Mol. Pharmacol. 69 (2006) 1820–1828. [19] M.L. Ponce, H.K. Kleinman, Identification of redundant angiogenic sites in laminin alpha1 and gamma1 chains, Exp. Cell Res. 285 (2003) 189–195. [20] A.G. Coombes, M.K. Yeh, E.C. Lavelle, S.S. Davis, The control of protein release from poly(DL-lactide co-glycolide) microparticles by variation of the external aqueous phase surfactant in the water-in oil-in water method, J. Control. Release 52 (1998) 311–320. [21] J. Panyam, V. Labhasetwar, Dynamics of endocytosis and exocytosis of poly(D,Llactide-co-glycolide) nanoparticles in vascular smooth muscle cells, Pharm. Res. 20 (2003) 212–220. [22] W.K. Lee, J.Y. Park, S. Jung, C.W. Yang, W.U. Kim, H.Y. Kim, et al., Preparation and characterization of biodegradable nanoparticles entrapping immunodominant peptide conjugated with PEG for oral tolerance induction, J. Control. Release 105 (2005) 77–88. [23] J.L. Edelman, M.R. Castro, Quantitative image analysis of laser-induced choroidal neovascularization in rat, Exp. Eye Res. 71 (2000) 523–533. [24] I. Semkova, S. Peters, G. Welsandt, H. Janicki, J. Jordan, U. Schraermeyer, Investigation of laser-induced choroidal neovascularization in the rat, Invest. Ophthalmol. Vis. Sci. 44 (2003) 5349–5354. [25] C. Martinez-Sancho, R. Herrero-Vanrell, S. Negro, Poly (D,L-lactide-co-glycolide) microspheres for long-term intravitreal delivery of aciclovir: influence of fatty and non-fatty additives, J. Microencapsul 20 (2003) 799–810. [26] Y. de Kozak, K. Andrieux, H. Villarroya, C. Klein, B. Thillaye-Goldenberg, M.C. Naud, et al., Intraocular injection of tamoxifen-loaded nanoparticles: a new treatment of experimental autoimmune uveoretinitis, Eur. J. Immunol. 34 (2004) 3702–3712. [27] Y. He, J.C. Wang, Y.L. Liu, Z.Z. Ma, X.A. Zhu, Q. Zhang, Therapeutic and toxicological evaluations of cyclosporine a microspheres as a treatment vehicle for uveitis in rabbits, J. Ocular Pharmacol. Ther. 22 (2006) 121–131.
293
[28] T.L. Jackson, R.J. Antcliff, J. Hillenkamp, J. Marshall, Human retinal molecular weight exclusion limit and estimate of species variation, Invest. Ophthalmol. Vis. Sci. 44 (2003) 2141–2146. [29] J. Mordenti, R.A. Cuthbertson, N. Ferrara, K. Thomsen, L. Berleau, V. Licko, et al., Comparisons of the intraocular tissue distribution, pharmacokinetics, and safety of 125I-labeled full-length and Fab antibodies in rhesus monkeys following intravitreal administration, Toxicol. Pathol. 27 (1999) 536–544. [30] C. Durairaj, J.C. Shah, S. Senapati, U.B. Kompella, Prediction of vitreal half-life based on drug physicochemical properties: quantitative structure–pharmacokinetic relationships (QSPKR), Pharm. Res. 26 (2009) 1236–1260. [31] S.L. Fialho, A. da Silva Cunha, Manufacturing techniques of biodegradable implants intended for intraocular application, Drug Deliv. 12 (2005) 109–116. [32] Y. Morita, A. Ohtori, M. Kimura, K. Tojo, Intravitreous delivery of dexamethasone sodium m-sulfobenzoate from poly(DL-lactic acid) implants, Biol. Pharm. Bull. 21 (1998) 188–190. [33] T. Moritera, Y. Ogura, Y. Honda, R. Wada, S.H. Hyon, Y. Ikada, Microspheres of biodegradable polymers as a drug-delivery system in the vitreous, Invest. Ophthalmol. Vis. Sci. 32 (1991) 1785–1790. [34] J.L. Bourges, S.E. Gautier, F. Delie, R.A. Bejjani, J.C. Jeanny, R. Gurny, et al., Ocular drug delivery targeting the retina and retinal pigment epithelium using polylactide nanoparticles, Invest. Ophthalmol. Vis. Sci. 44 (2003) 3562–3569. [35] E. Solheim, B. Sudmann, G. Bang, E. Sudmann, Biocompatibility and effect on osteogenesis of poly(ortho ester) compared to poly(DL-lactic acid), J. Biomed. Mater. Res. 49 (2000) 257–263. [36] A.C. Grayson, G. Voskerician, A. Lynn, J.M. Anderson, M.J. Cima, R. Langer, Differential degradation rates in vivo and in vitro of biocompatible poly(lactic acid) and poly(glycolic acid) homo- and co-polymers for a polymeric drugdelivery microchip, J. Biomater. Sci. Polym. Ed. 15 (2004) 1281–1304. [37] E. Sakurai, H. Ozeki, N. Kunou, Y. Ogura, Effect of particle size of polymeric nanospheres on intravitreal kinetics, Ophthalmic Res. 33 (2001) 31–36. [38] T. Pannicke, O. Uckermann, I. Iandiev, P. Wiedemann, A. Reichenbach, A. Bringmann, Ocular inflammation alters swelling and membrane characteristics of rat Muller glial cells, J. Neuroimmunol. 161 (2005) 145–154. [39] K.S. Soppimath, T.M. Aminabhavi, A.R. Kulkarni, W.E. Rudzinski, Biodegradable polymeric nanoparticles as drug delivery devices, J. Control. Release 70 (2001) 1–20. [40] R. Gref, P. Couvreur, G. Barratt, E. Mysiakine, Surface-engineered nanoparticles for multiple ligand coupling, Biomaterials 24 (2003) 4529–4537. [41] X. Chen, Multimodality imaging of tumor integrin alphavbeta3 expression, MiniRev. Med. Chem. 6 (2006) 227–234. [42] M. Yasuda, M. Ohbayashi, K. Ohhinata, T. Yamamoto, The involvement of integrin alphavbeta3 in polymorphonuclear leukocyte-induced angiogenesis in bovine aortic endothelial cells, Life Sci. 75 (2004) 421–434. [43] J.S. Kerr, S.A. Mousa, A.M. Slee, Alpha(v)beta(3) integrin in angiogenesis and restenosis, Drug News Perspect. 14 (2001) 143–150. [44] M.A. Speicher, R.P. Danis, M. Criswell, L. Pratt, Pharmacologic therapy for diabetic retinopathy, Expert Opin. Emerg. Drugs 8 (2003) 239–250. [45] A. Capello, E.P. Krenning, B.F. Bernard, W.A. Breeman, J.L. Erion, M. de Jong, Anticancer activity of targeted proapoptotic peptides, J. Nucl. Med. 47 (2006) 122–129. [46] T. Yasukawa, S. Hoffmann, W. Eichler, U. Friedrichs, Y.S. Wang, P. Wiedemann, Inhibition of experimental choroidal neovascularization in rats by an alpha(v)integrin antagonist, Curr. Eye Res. 28 (2004) 359–366. [47] H. Kim, S.B. Robinson, K.G. Csaky, Investigating the movement of intravitreal human serum albumin nanoparticles in the vitreous and retina, Pharm. Res. 26 (2009) 329–337. [48] H. Nishihara, Studies on the ultrastructure of the inner limiting membrane of the retina—distribution of anionic sites in the inner limiting membrane of the retina, Nippon Ganka Gakkai Zasshi 95 (1991) 951–958. [49] A.H. Bunt-Milam, J.C. Saari, I.B. Klock, G.G. Garwin, Zonulae adherentes pore size in the external limiting membrane of the rabbit retina, Invest. Ophthalmol. Vis. Sci. 26 (1985) 1377–1380. [50] R.A. Bejjani, D. BenEzra, H. Cohen, J. Rieger, C. Andrieu, J.C. Jeanny, et al., Nanoparticles for gene delivery to retinal pigment epithelial cells, Mol. Vis. 11 (2005) 124–132.
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Journal of Controlled Release 142 (2010) 286–293
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Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l
Nanoparticle–integrin antagonist C16Y peptide treatment of choroidal neovascularization in rats Hyuncheol Kim a,⁎, Karl G. Csaky b a b
Chemical and Biomolecular Engineering, Sogang University, #1 Shinsu-dong Mapo-gu, Seoul 121-742, Republic of Korea Department of Ophthalmology, Duke University Medical Center, Durham, North Carolina, USA
a r t i c l e
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Article history: Received 28 July 2009 Accepted 29 October 2009 Available online 4 November 2009 Keywords: Choroidal neovascularization Nanoparticles Integrin antagonist Intravitreal injection
a b s t r a c t Choroidal neovascularization (CNV) is the major cause of severe vision loss in patients with age-related macular degeneration (AMD). Present drug delivery may be limited by poor delivery to the choroid where CNV originates. The goal of this study was to develop a drug delivery system to deliver an integrin– antagonist peptide to the sub-retinal space. We developed polylactic acid/polylactic acid–polyethylene oxide nanoparticles (PLA/PLA-PEO) encapsulating the water-soluble integrin–antagonist peptide, C16Y (C16Y-NP). The PLA/PLA-PEO nanoparticles were 302 +/− 85.1 nm in size and demonstrated a two-week sustained release, in vitro, of encapsulated C16Y. Injected nanoparticles did not demonstrate retinal toxicity as determined by histopathology. C16Y peptide solution or C16Y-NP was injected 5 or 9 days post laser photocoagulation. A single intravitreal injection of C16Y peptide and C16Y-NP solution at both 5 days and 9 days post laser photocoagulation statistically inhibited CNV (p b 0.05). However, for the day 5 injections the area of choroidal neovascularization on day 12 was smaller for C16Y-NP than for C16Y peptide solution (p b 0.05) because of the short vitreous half-life of C16Y peptide solution. These results demonstrate the importance of sustained release delivery for the treatment of choroidal neovascularization associated with age-related macular degeneration. The intravitreally administered PLA/PLA-PEO containing coumarin was found to penetrate the retina and localize to the RPE. These results suggest that nanoparticles of biodegradable polymers may be a potential useful delivery system for intravitreal injection of drugs in the treatment of AMD. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Choroidal neovascularization (CNV) is the major cause of irreversible vision loss associated with age-related macular degeneration (AMD) [1]. Several therapies such as laser photocoagulation, photodynamic therapy, macular translocation and surgical removal are available to treat AMD [2]. However, these therapies usually fail because of CNV recurrence. Therefore, pharmacologic therapy has been developed to inhibit CNV. However, as CNV originates from the choroid, delivering therapeutic agents to the choroid and the retinal pigment epithelium to inhibit CNV is challenging because of several formidable barriers. Barriers for topical drug administration include tear film washout, physical properties of the cornea, the long diffusion distance between the cornea and retina and the rapid clearance by aqueous humor flow [3,4]. The transscleral drug delivery route is also limited by several active episcleral clearance mechanisms [5,6]. Indeed, placement of the anti-angiogenic agent anecortave acetate onto the sclera for the treatment of CNV was shown not to be effective
⁎ Corresponding author. Tel.: +82 2 705 8922; fax: +82 2 3273 0331. E-mail address: [email protected] (H. Kim). 0168-3659/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2009.10.031
[7]. Directly injecting drugs into the vitreous has shown favorable results, in multiple clinical trials, in delivering therapeutic agents to the posterior segment of the eye [8–10]. However, intravitreal injections invariably need to be repeated on a frequent basis and this approach is associated with potential serious complications including vitreous hemorrhage, retinal detachment, and endophthalmitis [11,12]. Therefore, sustained release intravitreal injectable delivery methods such as injected bioerodible or nonbioerodible systems have been developed [13,14] but do not address the issue of retinal penetration. The integrin heterodimers, αvβ3, αvβ5, and α5β1, play important roles in the pathogenesis of angiogenesis [15,16]. CNV predominantly expresses integrin αvβ3 and blocking this integrin heterodimer has been reported to inhibit neovascularization [17,18]. The scrambled C16Y peptide has been previously shown to block both the αvβ3 and α5β1 integrins and reduce chick chorioallantoic membrane angiogenesis and tumor growth [19]. Because peptides typically have very short intravitreal half-lives, in the present study we have developed biodegradable polymer-based nanoparticles to deliver the angiogenesis inhibitor peptide, C16Y, to the posterior segment of the eye. The aim of the study was to obtain a more sustained delivery of the peptide and improve retinal penetration of the drug.
2. Materials and methods 2.1. Materials Poly(D,L-lactide) (PLA) was purchased from Birmingham Polymers (Birmingham, AL, USA). Poly(ethylene oxide-b-lactide) (DL form) (PEO(700)-b-PLA(3750)) was purchased from Polymer Source (Polymer Source Inc., Dorval, Quebec). C16Y peptide (DFKLFAVYIKYR) was synthesized by Celtek Peptide (Celtek Bioscience, LLC., Nashville, TN, USA). Polyvinyl alcohol (PVA, 88% mol hydrolyzed, MW 31,000) was obtained from Fluka (Sigma-Aldrich Corp., St. Louis, MO, USA). All other chemicals were of reagent grade. 2.2. Nanoparticle formulation The nanoparticle preparation method was a modification of the W1/O/W2 emulsion method previously described [20]. Briefly, 5.34 mg of C16Y peptide was dissolved in 0.3 mL of formamide. 50.0 mg of PLA was dissolved in 2 mL of methylene chloride. 50 µL of PLA-PEO acetone solution (0.1 g/mL) was added to the PLA organic solution. 0.3 mL of C16Y peptide solution (17.8 mg/mL) was added and emulsified in the PLA and PLA-PEO organic solution by sonicating with 20 W for 30 s. 10 mL of 5% (w/v) PVA solution was added to the first emulsion and then the solution was sonicated with 20 W for 30 s. This emulsion was gently poured into 50 mL of a 0.5% (w/v) PVA solution with magnetic bar stirring. Then, the emulsion was stirred overnight to evaporate the organic solvents. Nanoparticles were recovered by centrifugation at 16,000 RPM for 40 min and washed with ultrapure water. This process was repeated three times to remove residual PVA and organic solvents and the nanoparticles were freeze-dried. To determine the distribution of intravitreally administered PLA/ PLA-PEO nanoparticles in the retina, a lipophilic fluorescent marker, 6-coumarin, was incorporated in the nanoparticles. The dye was reported to associate with the polymer matrix of nanoparticles and not leach from the nanoparticles [21]. The fluorescence dye, coumarin (0.2 mg) was dissolved in 50.0 mg of PLA organic solution and coumarin encapsulating PLA/PLA-PEO nanoparticles were fabricated as described above. 2.3. Particle size analysis Particle size was determined by photon correlation spectroscopy (PCS) using quasi-elastic light scattering equipment (ZetaPlus™ equipped with particle sizing mode, Brookhaven Instrument Corp., Holtsville, NY, USA). A dilute suspension of nanoparticles was prepared in ultrapure water. The sample was subjected to particle size analysis and analyzed in duplicate with 10 readings per nanoparticle sample. Zeta potential of nanoparticles in distilled water was determined using ZetaPlus™ in the zeta potential analysis mode in duplicate with 10 readings per nanoparticle sample. Morphological evaluation of the nanoparticles was performed using scanning electron microscopy (SEM) (Hitachi S-4500). 2.4. In vitro release study In vitro release of C16Y from nanoparticles was determined as previously described [22]. 18.0 mg of nanoparticles (containing 1.07 mg of C16Y peptide) were placed in a conical tube containing 1.5 mL of ultrapure water and incubated at 37 °C under continuous shaking. At various time intervals, the sample was centrifuged at 16,000 RPM for 40 min and 0.7 mL of the supernatant was analyzed by HPLC. The peptide release study was continued after replacement with the same volume of fresh ultrapure water and resuspension by vortexing. In vitro release rates from the nanoparticles were determined by assaying the peptide concentrations over time with a
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reverse-phase high-performance liquid chromatography (HPLC) assay. Samples or standards with volumes of 5 to 200 µL were injected with an autosampler (model G1329A; Agilent Technologies, Palo Alto, CA, USA). A 250 × 4.6 mm (5 µm) C18 polymeric column (Vydac, Hesperia, CA, USA) was used for separation. Separation was conducted by 1.0 mL/min isocratic elution of water with 0.1% TFA and acetonitrile with 0.1% TFA by volume and a pump (model G1312A; Agilent). The concentrations of the samples were monitored at 230 nm with a UV detector (model G115A; Agilent) and analyzed on computer (Chemstation software; Agilent). The retention time was 8.1 min. The standard curve was linear (R2 = 0.99) over the range of 100 to 75,000 ng/mL. The drug detection limit for the peptide in solvent was 10 ng/mL. Peptide release profiles were generated for each nanoparticle formulation in terms of cumulative peptide release versus time. 2.5. Argon laser-induced CNV in rats All animal studies conformed to the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. Male brown Norway rats (N = 18) aging 8–12 weeks were anesthetized by intramuscular injection of ketamine (70 mg/kg) and xylazine (30 mg/kg) and both pupils were dilated with 1% Tropicamide (Mydriacyl®, Alcon, Fort Worth, TX, USA). Hydroxypropyl methycellulose (DECA Pharmaceuticals, LLC., Bowling Green, KY, USA) was applied to each eye and a microscope cover glass was used as a contact lens. Both eyes received four laser burns between retinal vessels around the optic nerve head using the blue-green setting of a Lumenis Ultima Argon Laser (Lumenis Inc., New York, NY, USA) using a laser power of 180 mW for 0.1 s and a spot size of 50 μm. 2.6. Effect of intravitreal C16Y encapsulating nanoparticle on experimental CNV Laser-induced CNV has been demonstrated to grow in size reaching a maximum area at 2 weeks after laser treatment. On day 0, laser photocoagulation was performed and either on day 5 (n = 6—each group) or day 9 (n = 3—each group), 10 μL of PBS alone or PBS containing empty nanoparticles (0.12 mg/μL), C16Y (3 μg/μL), or C16YNP (0.12 mg/μL) was injected. The materials were injected intravitreally with a 32-gauge needle after topical anesthesia with 1% tropicamide ophthalmic solution (Mydriacyl®, Alcon, Forth Worth, Texas, USA). On day 12, the animals were euthanized by CO2 inhalation. 2.7. Visualization and quantification of CNV Blood vessels were visualized using fluorescent isothiocyanate (FITC)-dextran (MW = 2 × 106 Da) perfusion and preparation of choroidal flat mounts as described previously [23,24]. Briefly, the eyes were sectioned at the equator and the anterior segment of the eye, vitreous and retina were removed. The posterior segment of the eye including the sclera and the choroid, was dissected into quarters by four radial cuts and mounted on a microscope slide with a fluorescent mounting media (Vectashield®, Vector Laboratories, Inc., Burlingame, CA, USA). Images of the flat mounts were captured with a fluorescent microscope (DM5000B, Leica Microsystems Inc, Bannockburn, IL, USA) using a FITC filter (excitation 471 nm and emission 503 nm) and a CCD camera coupled to a computer with image analysis software (Volocity 3.6.1, ImproVision, Inc., Lexington, MA). Blood vessels within the neovascular lesion that were labeled with FITC-dextran were highlighted and converted into a binary image with ImageJ 1.34S (National Institutes of Health, Bethesda, MD, USA). The area occupied by black pixels was calculated with Matlab software (version 6.5.1, The MathWorks, Inc., Natick, MA, USA).
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2.8. Determination of the retinal distribution of intravitreally administered PLA/PLA-PEO nanoparticles 10 μL of a suspension of PLA/PLA-PEO nanoparticles encapsulating coumarin dye (18.5 μg/μL) was injected into the vitreous as described above. The eyes were enucleated and frozen in the freezing mold with optimal cutting temperature (O.C.T) compound 6 h, 1 day, and 3 days post intravitreal injection. Then, the eyes were kept at − 20 °C overnight and 10 μm sections were generated. The distribution of nanoparticles in the retina was examined under an epifluorescence microscope (BX50; Olympus, Tokyo, Japan). 2.9. Toxicity of nanoparticle Nanoparticles were manufactured as described above. 10 μl of nanoparticles (0.12 mg/μL) were injected in the right eyes of three rats. Three days post injection, the eyes were enucleated and frozen immediately using the OCT compound. The frozen eyes were sectioned with a cryotome and then fixed with 4% PFA in 1× PBS buffer. The sectioned eyes were stained with hematoxylin and eosin for light microscopic examination. 2.10. Statistical analysis The differences in the mean area of choroidal neovascularization at each group from the control group were compared and tested by the analysis of variance (ANOVA) using PSI-Plot version 7.3 (Poly software International, Inc., Pearl River, NY, USA). Significant differences were determined for p b 0.05. 3. Results 3.1. Nanoparticle analysis and toxicity PLA/PLA-PEO nanoparticles containing C16Y peptide demonstrated a bimodal size distribution in two size ranges: 114–126 nm and 302–367 nm (Fig. 1A). The average zeta potential was measured to be −38.26 +/− 1.42 mV (mean +/− SE, n = 10) in ultrapure water (Fig. 1B). Fig. 1C demonstrates a consistent spherical shape of generated PLA/PLA-PEO nanoparticles as seen by scanning electron microscopy. The measured amount of non-encapsulated C16Y peptide in the supernatant during the purification was found to be 2.37 mg. The starting amount of C16Y peptide was 5.34 mg, giving an encapsulation efficiency of 55.6%. The release of C16Y from the PLA/PLA-PEO nanoparticles in pure water was measured in vitro at 37 °C. 19 mg of nanoparticles containing 1.07 mg of C16Y were placed into pure water. The nanoparticles exhibited sustained release of the encapsulated C16Y peptide over 6-weeks (Fig. 2). To test for retinal toxicity, retinal histology was performed following intravitreal injection of PLA/PLA-PEO. The treated eyes (Fig. 3A) showed no evidence of inflammatory cells or loss of normal retinal architecture, compared to the control eye (Fig. 3B). 3.2. Angiographic analysis of RPE–choroid–sclera flat mount Both C16Y peptide solution and C16Y-NP were evaluated with a rat laser model of CNV. High molecular weight FITC-dextran was perfused to visualize the choroidal neovascularization. Figs. 4 and 5 show the representative laser-induced CNV from each group on day 12 post laser photocoagulation following intravitreal injection on day 5 or day 9, respectively. There was no difference in the neovascular area between the PBS alone and PBS containing nanoparticle groups injected on day 5 (p N 0.05) and day 9 (p N 0.05) post laser photocoagulation, respectively
Fig. 1. Particle analysis and electron microscope photograph. The size and surface charge of C16Y encapsulating PLA/PLA-PEO nanoparticles were 302.5 ± 85.1 nm (A) and − 38.26 ± 1.42 mV (B), respectively. (C) The nanoparticles containing C16Y peptide exhibited a spherical shape.
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3.3. Retinal distribution of intravitreally administered PLA/PLA-PEO nanoparticles PLA/PLA-PEO nanoparticles encapsulating the fluorescence dye (coumarin) diffused into the outer plexiform layer by 6 h post intravitreal injection (Fig. 6A) and were endocytosed into the RPE by 24 h (Fig. 6B). Nanoparticles were still retained in the retina 3 days after intravitreal administration (Fig. 6C). The results demonstrated that PLA/PLA-PEO nanoparticles over 100-nm in size easily penetrate the retina structure into the RPE following intravitreal injection. 4. Discussion
Fig. 2. PLA/PLA-PEO nanoparticles release C16Y peptide over six weeks in vitro.
(Figs. 4E and 5E). Empty PLA/PLA-PEO nanoparticles injected intravitreally at either day 5 or day 9 was found to have no antiangiogenic activity. A single intravitreal injection of C16Y peptide and C16Y-NP solution at both 5 days and 9 days post laser photocoagulation statistically inhibited CNV (p b 0.05). However, C16Y-NP solution injected on day 5 reduced the area of choroidal neovascularization more than the C16Y peptide solution injected on day 5 (p b 0.05).
Fig. 3. Representative histopathologic sections through the retina region of (A) the rat eye three days post intravitreal injection of PLA/PLA-PEO nanoparticles and (B) the non-treated rat eye. No inflammatory response or toxicity to the photoreceptors was observed (stain, hematoxylin and eosin). GCL, IPL, INL, OPL, ONL, PRL, and RPE represent the ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, photoreceptor layer, and retinal pigment epithelium, respectively.
Intravitreally administered micro- or nano-sized particles were reported to be able to deliver therapeutic agents to the posterior segment of the eye for an extended period of time [25–27]. In this study, we evaluated the effect of intravitreal injection of PLA/PLA-PEO nanoparticles encapsulating the anti-angiogenic C16Y peptide on inhibition of experimental CNV. The intravitreal C16Y peptide could be expected to diffuse easily into the sub-retinal space following intravitreal injection because the peptide's molecular weight (MW = 1600 Da) is much smaller than the maximum size (around 80 kDa) of solutes, that are readily able to penetrate the retina structure [28,29]. Both the C16Y peptide and C16Y-NP solutions, which were injected intravitreally on days 5 and 9 post laser photocoagulation, inhibited the experimental CNV, compared to the control (p b 0.05). However, C16Y-NP injected on day 5 was found to inhibit the choroidal neovascularization dramatically more than the C16Y peptide solution on day 5 (p b 0.05). In the C16Y peptide solution, 30 µL of C16Y peptide was injected. On the other hand, 1.2 mg of C16Y-NP could be expected to deliver 1.16 µg of C16Y peptide for 7 days, according to the in vitro release study. Although the C16Y solution injection delivered 25-fold more peptide than that estimated for C16Y-NP injection, the choroidal neovascularization was inhibited less with the C16Y peptide solution (p b 0.05) (Fig. 4E). The elimination of intravitreally administered water-soluble C16Y peptide was so active that the half-life of intravitreal C16Y peptide solution was not long enough for the peptide injected on day 5 post laser photocoagulation to inhibit the experimental CNV as much as the C16Y-NP injected on day 5 did. This indicates the importance of sustained release delivery for the treatment of choroidal neovascularization associated with age-related macular degeneration. The elimination route from the vitreous can be by diffusion into the anterior and then drainage through the aqueous pathway or across the retina. The half-life of intravitreal C16Y can be estimated to be around 10 h from half-lives of other agents [30]. Most of the C16Y peptide administered intravitreally in solution might then be eliminated within two days, which could account for the lesser inhibition of experimental CNV than that observed following C16Y-NP administration. C16Y-NP delivered the therapeutic agent long enough to inhibit the experimental CNV following intravitreal injection on both day 5 and day 9 post laser photocoagulation (p b 0.05). These results demonstrate that biodegradable polymer-based nanoparticles may be a potential intravitreal drug delivery system for the treatment of choroidal neovascularization associated with age-related macular degeneration. However, the optimal release kinetics of the biodegradable polymer-based nanoparticles should be investigated with a modified polymer ratio before clinical application. Polylactic acid polymer has been investigated for ocular drug delivery because of biocompatibility [31,32]. Intravitreally administered PLA microparticles were non-toxic at ocular tissues [27,33]. On the other hand, intravitreally injected PLA nanoparticles induced mild inflammation at the retina at early time points [26,34]. PLA polymer is known to induce mild and chronic inflammatory responses in soft tissues [35,36]. Intravitreal microparticles remain suspended in the vitreous. Intravitreal nanoparticles, on the other hand, diffuse to the
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Fig. 4. Representative images of anterior segments dissected on day 12 post laser photocoagulation. Each image is centered about a 50 µm diameter laser spot region. Each group of animals received intravitreal injection on day 5 of: (A) PBS alone; (B) PBS containing empty PLA/PLA-PEO nanoparticles; (C) C16Y peptide solution; (D) C16Y peptide encapsulating PLA/PLA-PEO nanoparticles. (E) Image analysis demonstrates that a single intravitreal injection of C16Y peptide and C16Y-NP solution at both 5 days and 9 days post laser photocoagulation statistically inhibited CNV (p b 0.05). However, for injection on day 5, the area of choroidal neovascularization on day 12 was smaller for C16Y-NP than for C16Y solution (p b 0.05).
retina and accumulate on the inner limiting membrane with some particles diffusing deeper into the retina [37]. So, we hypothesized that the inflammation response of the PLA based nanoparticles came from direct interaction between hydrophobic PLA polymer and ocular tissues. Inflammation response has been known to negatively influence retina physiology [38]. Hydrogels, such as polyethylene
glycol (PEG) and polyethylene oxide (PEO), have been known to induce no inflammation response at soft tissues [39]. So, we blended polylactic acid with a co-polymer of PLA (MW = 700) and PEO (MW = 3750) to reduce the contact of naked PLA and retina tissue. PLA, the hydrophobic part of the co-polymer, is embedded into the organic phase; on the other hand, PEO, the hydrophilic part of the co-
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Fig. 5. Representative images of anterior segments dissected on day 12 post laser photocoagulation. Each image is centered about a 50 µm diameter laser spot region. Each group of animals received intravitreal injection on day 9 of: (A) PBS alone; (B) PBS containing empty PLA/PLA-PEO nanoparticles; (C) C16Y peptide solution; (D) C16Y peptide encapsulating PLA/PLA-PEO nanoparticles. (E) Image analysis demonstrates that both intravitreally injected C16Y-NP and C16Y peptide solution injected on day 9 inhibited the CNV significantly (p b 0.05) compared to the control.
polymer, is directed into the aqueous phase around the PLA organic droplets during the nanoparticle fabrication [40]. The toxicity study showed no inflammatory response from PLA/PLA-PEO nanoparticles in the retinal tissue on day 3 post intravitreal injection (Fig. 3). In the current study, we could reduce the toxicity of a PLA nanoparticle by decreasing the direct contact of PLA and retinal tissue with PEO. However, the intravitreally administered PLA/PLA-PEO nanoparticles degrade with time to increase the contact of PLA and retinal tissue.
The increased contact might induce an inflammatory response in the ocular tissues at a later time than for the observations in the present study. The evaluation of long-term toxicity should be performed before clinical trials and remains for future work. The αvβ3 integrin is known to play an important role in angiogenesis and to express predominantly in neovascular ocular tissues in age-related macular degeneration patients [15,16,41–43]. Blocking the ligation of integrin into the extracellular matrix has been
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Recently, nano-sized drug delivery carriers have been intensively investigated for delivering therapeutic agents to the posterior segment of the eye [13]. However, the information on the distribution of intravitreally administered nanoparticles is limited. Upon intravitreal administration, many nanoparticles first settled down on the inner limiting membrane (ILM), which might be the major barrier to the penetration of intravitreal nanoparticles into the retina [34]. And then, some nanoparticles passed through the ILM penetrating deeper retina structures and were observed in the RPE for several months [26,34,47]. Some retina layers show formidable barriers against the trans-retinal penetration of macromolecules. For example, the ILM is composed of a fine three-dimensional meshwork structure with numerous pores whose size ranges from 10 to 25 nm [48]. And the external limiting membrane (ELM) is formed by rows of zonulae adherents, whose pore radius is between 30 and 36 Å [49]. However, this study demonstrated that intravitreally administered PLA/PLAPEO nanoparticles greater than 100 nm in diameter penetrate the retinal structures (Fig. 6). Our previous study demonstrated that the Müller cells might play an important role in the retinal penetration of human serum albumin nanoparticles [47]. So, we surmise that PLA/ PLA-PEO nanoparticles penetrate through the Müller cells. However, the verification of the trans-retinal penetration mechanism of the PLA/PLA-PEO nanoparticles was beyond the scope of this study. Sakurai et al. [37] reported that intravitreally administered polystyrene nanoparticles were distributed homogeneously and the half-lives of intravitreal 2 μm, 200 nm, or 50 nm polystyrene were 5.4, 8.6, and 10.1 days, respectively. We found in the current study that the intravitreally administered PLA/PLA-PEO nanoparticles dispersed in the vitreous and thus might cause vision discomfort to patients. In addition, many of the nanoparticles were found in the ciliary body and the trabecular meshwork. The nanoparticles are able to enter cells easily by endocytosis and pinocytosis and then are degraded into biocompatible monomer [50]. Nanoparticles inside the cells may induce disturbance of the aqueous humor circulations. Although the PLA/PLA-PEO nanoparticles did not provoke inflammation, the interaction between endocytosed nanoparticles and ocular cells must be investigated intensively before clinical application. 5. Conclusions In conclusion, we developed non-toxic ocular nanoparticles by blending PLA and PLA-PEO co-polymer together to deliver the watersoluble integrin–antagonist peptide, C16Y, to the sub-retinal space in order to inhibit the experimental choroidal neovascularization. The nanoparticle based C16Y peptide delivery was superior to C16Y peptide solution delivery. Fig. 6. The retinal distribution of intravitreally administered coumarin encapsulating PLA/PLA-PEO nanoparticles (A) 6 h; (B) 1 day; (C) 3 days post injection. (B) Inset demonstrates the endocytosis of the nanoparticles in the RPE. GCL, IPL, INL, OPL, ONL, PRL, and RPE represent the ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, photoreceptor layer, and retinal pigment epithelium, respectively. All images were captured at × 20 magnification (inset at × 60).
proven to prevent blood vessel formation in several in vivo models [15,44]. RGD motif peptides target and block αvβ3 [2,45,46]. The intravitreal injection of 200 μg or 100 μg RGD peptide 9 days and 11 days post laser photocoagulation inhibited choroidal neovascularization efficiently. However, the intravitreal injection of 50 μg RGD peptide did not inhibit the CNV, compared to the 0 μg control injection. On the other hand, the intravitreal injection of 30 μg of C16Y peptide on 9 days post laser photocoagulation showed statistically significant difference from the PBS alone injection control group. Therefore, the C16Y peptide appears to be more efficient at inhibiting CNV than RGD based peptide.
Acknowledgements The work is supported in part by Sogang University internal research grant (200910005.01). The authors are grateful to Dr. Peter M. Bungay, National Institutes of Health, for his kind help and useful suggestions. References [1] F.L. Ferris 3rd, S.L. Fine, L. Hyman, Age-related macular degeneration and blindness due to neovascular maculopathy, Arch. Ophthalmol. 102 (1984) 1640–1642. [2] S.L. Fine, J.W. Berger, M.G. Maguire, A.C. Ho, Age-related macular degeneration, N. Engl. J. Med. 342 (2000) 483–492. [3] C.L. Bourlais, L. Acar, H. Zia, P.A. Sado, T. Needham, R. Leverge, Ophthalmic drug delivery systems—recent advances, Prog. Retin. Eye Res. 17 (1998) 33–58. [4] A.A. Moshfeghi, G.A. Peyman, Micro- and nanoparticulates, Adv. Drug Deliv. Rev. 57 (2005) 2047–2052. [5] H. Kim, M.R. Robinson, M.J. Lizak, G. Tansey, R.J. Lutz, P. Yuan, et al., Controlled drug release from an ocular implant: an evaluation using dynamic three-dimensional magnetic resonance imaging, Invest. Ophthalmol. Vis. Sci. 45 (2004) 2722–2731.
[6] M.R. Robinson, S.S. Lee, H. Kim, S. Kim, R.J. Lutz, C. Galban, et al., A rabbit model for assessing the ocular barriers to the transscleral delivery of triamcinolone acetonide, Exp. Eye Res. 82 (2006) 479–487. [7] J.S. Slakter, T.W. Bochow, D.J. D'Amico, B. Marks, J. Jerdan, E.K. Sullivan, et al., Anecortave acetate (15 milligrams) versus photodynamic therapy for treatment of subfoveal neovascularization in age-related macular degeneration, Ophthalmology 113 (2006) 3–13. [8] D.M. Brown, P.K. Kaiser, M. Michels, G. Soubrane, J.S. Heier, R.Y. Kim, et al., Ranibizumab versus verteporfin for neovascular age-related macular degeneration, N. Engl. J. Med. 355 (2006) 1432–1444. [9] E.S. Gragoudas, A.P. Adamis, E.T. Cunningham Jr, M. Feinsod, D.R. Guyer, VEGF inhibition study in ocular neovascularization clinical trial group. , Pegaptanib for neovascular age-related macular degeneration, N. Engl. J. Med. 351 (2004) 2805–2816. [10] P.J. Rosenfeld, D.M. Brown, J.S. Heier, D.S. Boyer, P.K. Kaiser, C.Y. Chung, et al., Ranibizumab for neovascular age-related macular degeneration, N. Engl. J. Med. 355 (2006) 1419–1431. [11] J.I. Lim, R.A. Wolitz, A.H. Dowling, H.R. Bloom, A.R. Irvine, D.M. Schwartz, Visual and anatomic outcomes associated with posterior segment complications after ganciclovir implant procedures in patients with AIDS and cytomegalovirus retinitis, Am. J. Ophthalmol. 127 (1999) 288–293. [12] T.S. Shane, D.F. Martin, Endophthalmitis after ganciclovir implant in patients with AIDS and cytomegalovirus retinitis, Am. J. Ophthalmol. 136 (2003) 649–654. [13] R.C. Nagarwal, S. Kant, P.N. Singh, P. Maiti, J.K. Pandit, Polymeric nanoparticulate system: a potential approach for ocular drug delivery, J. Control. Release 136 (2009) 2–13. [14] K. Hironaka, Y. Inokuchi, Y. Tozuka, M. Shimazawa, H. Hara, H. Takeuchi, Design and evaluation of a liposomal delivery system targeting the posterior segment of the eye, J. Control. Release 136 (2009) 247–253. [15] P.C. Brooks, R.A. Clark, D.A. Cheresh, Requirement of vascular integrin alpha v beta 3 for angiogenesis, Science 264 (1994) 569–571. [16] M. Friedlander, C.L. Theesfeld, M. Sugita, M. Fruttiger, M.A. Thomas, S. Chang, et al., Involvement of integrins alpha v beta 3 and alpha v beta 5 in ocular neovascular diseases, Proc. Natl Acad. Sci. USA 93 (1996) 9764–9769. [17] H. Kamizuru, H. Kimura, T. Yasukawa, Y. Tabata, Y. Honda, Y. Ogura, Monoclonal antibody-mediated drug targeting to choroidal neovascularization in the rat, Invest. Ophthalmol. Vis. Sci. 42 (2001) 2664–2672. [18] N. Umeda, S. Kachi, H. Akiyama, G. Zahn, D. Vossmeyer, R. Stragies, et al., Suppression and regression of choroidal neovascularization by systemic administration of an {alpha}5{beta}1 integrin antagonist, Mol. Pharmacol. 69 (2006) 1820–1828. [19] M.L. Ponce, H.K. Kleinman, Identification of redundant angiogenic sites in laminin alpha1 and gamma1 chains, Exp. Cell Res. 285 (2003) 189–195. [20] A.G. Coombes, M.K. Yeh, E.C. Lavelle, S.S. Davis, The control of protein release from poly(DL-lactide co-glycolide) microparticles by variation of the external aqueous phase surfactant in the water-in oil-in water method, J. Control. Release 52 (1998) 311–320. [21] J. Panyam, V. Labhasetwar, Dynamics of endocytosis and exocytosis of poly(D,Llactide-co-glycolide) nanoparticles in vascular smooth muscle cells, Pharm. Res. 20 (2003) 212–220. [22] W.K. Lee, J.Y. Park, S. Jung, C.W. Yang, W.U. Kim, H.Y. Kim, et al., Preparation and characterization of biodegradable nanoparticles entrapping immunodominant peptide conjugated with PEG for oral tolerance induction, J. Control. Release 105 (2005) 77–88. [23] J.L. Edelman, M.R. Castro, Quantitative image analysis of laser-induced choroidal neovascularization in rat, Exp. Eye Res. 71 (2000) 523–533. [24] I. Semkova, S. Peters, G. Welsandt, H. Janicki, J. Jordan, U. Schraermeyer, Investigation of laser-induced choroidal neovascularization in the rat, Invest. Ophthalmol. Vis. Sci. 44 (2003) 5349–5354. [25] C. Martinez-Sancho, R. Herrero-Vanrell, S. Negro, Poly (D,L-lactide-co-glycolide) microspheres for long-term intravitreal delivery of aciclovir: influence of fatty and non-fatty additives, J. Microencapsul 20 (2003) 799–810. [26] Y. de Kozak, K. Andrieux, H. Villarroya, C. Klein, B. Thillaye-Goldenberg, M.C. Naud, et al., Intraocular injection of tamoxifen-loaded nanoparticles: a new treatment of experimental autoimmune uveoretinitis, Eur. J. Immunol. 34 (2004) 3702–3712. [27] Y. He, J.C. Wang, Y.L. Liu, Z.Z. Ma, X.A. Zhu, Q. Zhang, Therapeutic and toxicological evaluations of cyclosporine a microspheres as a treatment vehicle for uveitis in rabbits, J. Ocular Pharmacol. Ther. 22 (2006) 121–131.
293
[28] T.L. Jackson, R.J. Antcliff, J. Hillenkamp, J. Marshall, Human retinal molecular weight exclusion limit and estimate of species variation, Invest. Ophthalmol. Vis. Sci. 44 (2003) 2141–2146. [29] J. Mordenti, R.A. Cuthbertson, N. Ferrara, K. Thomsen, L. Berleau, V. Licko, et al., Comparisons of the intraocular tissue distribution, pharmacokinetics, and safety of 125I-labeled full-length and Fab antibodies in rhesus monkeys following intravitreal administration, Toxicol. Pathol. 27 (1999) 536–544. [30] C. Durairaj, J.C. Shah, S. Senapati, U.B. Kompella, Prediction of vitreal half-life based on drug physicochemical properties: quantitative structure–pharmacokinetic relationships (QSPKR), Pharm. Res. 26 (2009) 1236–1260. [31] S.L. Fialho, A. da Silva Cunha, Manufacturing techniques of biodegradable implants intended for intraocular application, Drug Deliv. 12 (2005) 109–116. [32] Y. Morita, A. Ohtori, M. Kimura, K. Tojo, Intravitreous delivery of dexamethasone sodium m-sulfobenzoate from poly(DL-lactic acid) implants, Biol. Pharm. Bull. 21 (1998) 188–190. [33] T. Moritera, Y. Ogura, Y. Honda, R. Wada, S.H. Hyon, Y. Ikada, Microspheres of biodegradable polymers as a drug-delivery system in the vitreous, Invest. Ophthalmol. Vis. Sci. 32 (1991) 1785–1790. [34] J.L. Bourges, S.E. Gautier, F. Delie, R.A. Bejjani, J.C. Jeanny, R. Gurny, et al., Ocular drug delivery targeting the retina and retinal pigment epithelium using polylactide nanoparticles, Invest. Ophthalmol. Vis. Sci. 44 (2003) 3562–3569. [35] E. Solheim, B. Sudmann, G. Bang, E. Sudmann, Biocompatibility and effect on osteogenesis of poly(ortho ester) compared to poly(DL-lactic acid), J. Biomed. Mater. Res. 49 (2000) 257–263. [36] A.C. Grayson, G. Voskerician, A. Lynn, J.M. Anderson, M.J. Cima, R. Langer, Differential degradation rates in vivo and in vitro of biocompatible poly(lactic acid) and poly(glycolic acid) homo- and co-polymers for a polymeric drugdelivery microchip, J. Biomater. Sci. Polym. Ed. 15 (2004) 1281–1304. [37] E. Sakurai, H. Ozeki, N. Kunou, Y. Ogura, Effect of particle size of polymeric nanospheres on intravitreal kinetics, Ophthalmic Res. 33 (2001) 31–36. [38] T. Pannicke, O. Uckermann, I. Iandiev, P. Wiedemann, A. Reichenbach, A. Bringmann, Ocular inflammation alters swelling and membrane characteristics of rat Muller glial cells, J. Neuroimmunol. 161 (2005) 145–154. [39] K.S. Soppimath, T.M. Aminabhavi, A.R. Kulkarni, W.E. Rudzinski, Biodegradable polymeric nanoparticles as drug delivery devices, J. Control. Release 70 (2001) 1–20. [40] R. Gref, P. Couvreur, G. Barratt, E. Mysiakine, Surface-engineered nanoparticles for multiple ligand coupling, Biomaterials 24 (2003) 4529–4537. [41] X. Chen, Multimodality imaging of tumor integrin alphavbeta3 expression, MiniRev. Med. Chem. 6 (2006) 227–234. [42] M. Yasuda, M. Ohbayashi, K. Ohhinata, T. Yamamoto, The involvement of integrin alphavbeta3 in polymorphonuclear leukocyte-induced angiogenesis in bovine aortic endothelial cells, Life Sci. 75 (2004) 421–434. [43] J.S. Kerr, S.A. Mousa, A.M. Slee, Alpha(v)beta(3) integrin in angiogenesis and restenosis, Drug News Perspect. 14 (2001) 143–150. [44] M.A. Speicher, R.P. Danis, M. Criswell, L. Pratt, Pharmacologic therapy for diabetic retinopathy, Expert Opin. Emerg. Drugs 8 (2003) 239–250. [45] A. Capello, E.P. Krenning, B.F. Bernard, W.A. Breeman, J.L. Erion, M. de Jong, Anticancer activity of targeted proapoptotic peptides, J. Nucl. Med. 47 (2006) 122–129. [46] T. Yasukawa, S. Hoffmann, W. Eichler, U. Friedrichs, Y.S. Wang, P. Wiedemann, Inhibition of experimental choroidal neovascularization in rats by an alpha(v)integrin antagonist, Curr. Eye Res. 28 (2004) 359–366. [47] H. Kim, S.B. Robinson, K.G. Csaky, Investigating the movement of intravitreal human serum albumin nanoparticles in the vitreous and retina, Pharm. Res. 26 (2009) 329–337. [48] H. Nishihara, Studies on the ultrastructure of the inner limiting membrane of the retina—distribution of anionic sites in the inner limiting membrane of the retina, Nippon Ganka Gakkai Zasshi 95 (1991) 951–958. [49] A.H. Bunt-Milam, J.C. Saari, I.B. Klock, G.G. Garwin, Zonulae adherentes pore size in the external limiting membrane of the rabbit retina, Invest. Ophthalmol. Vis. Sci. 26 (1985) 1377–1380. [50] R.A. Bejjani, D. BenEzra, H. Cohen, J. Rieger, C. Andrieu, J.C. Jeanny, et al., Nanoparticles for gene delivery to retinal pigment epithelial cells, Mol. Vis. 11 (2005) 124–132.
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