Abstract
purpose. To develop a controlled-drug delivery system for the long-term inhibition of vascular endothelial growth factor (VEGF) and its mediated responses.
methods. Poly(lactic-co-glycolic)acid (PLGA) microspheres containing anti-VEGF RNA aptamer (EYE001) formulations in the solid-state were developed by an oil-in-oil solvent evaporation process. In vitro experiments were performed to characterize the release profiles. Stability and bioactivity of the released drug were assayed by monitoring the RNA aptamer’s ability to inhibit VEGF-induced cell proliferation in human umbilical vein endothelial cells (HUVECs). Cell proliferation experiments were conducted with aptamer aliquots collected after short-, mid-, and long-term release time points. To investigate the feasibility of this polymer device as a potential transscleral delivery device, an in vitro apparatus was developed to assess polymer hydration and degradation through rabbit sclera and subsequent delivery through it.
results. PLGA microspheres were able to deliver EYE001 in a sustained manner, with an average rate of 2 μg/d over a period of 20 days. Solid-state stabilization of the aptamer with disaccharide trehalose before lyophilization and encapsulation in PLGA rendered the drug more stable after release. Cell proliferation experiments demonstrated that the bioactivity of the aptamer was preserved after release, as indicated by inhibition of endothelial cell proliferation after incubation with VEGF. Microspheres packed into a sealed chamber and placed onto the “orbital” part of a rabbit sclera for a period of 6 days became hydrated and started to degrade, as shown by scanning electron microscopy (SEM). As a result, the aptamer was delivered from the microspheres through the sclera, as determined spectrophotometrically.
conclusions. The loading of aptamer-containing microspheres into a device and placing it on the orbital surface of the sclera was assessed and shown to be feasible. RNA aptamer EYE001 encapsulated in PLGA was delivered over a period of 20 days with retained activity. This method represents a promising approach for the transscleral delivery of drugs and the treatment of choroidal and retinal diseases.
Vascular endothelial growth factor (VEGF) has been identified as a key positive regulator of angiogenesis.
1 It acts as an endothelial cell mitogen and chemoattractant in vitro
2 3 and induces vascular permeability and angiogenesis in vivo.
2 3 Elevated VEGF expression is correlated with several forms of ocular neovascularization that often lead to severe vision loss, including diabetic retinopathy,
4 retinopathy of prematurity,
5 and macular degeneration.
6 Thus, agents that specifically inhibit VEGF may have great utility in combating a variety of human diseases for which few effective treatments are presently available.
7
Recently, a method used to isolate oligonucleotide ligands (aptamers) from libraries of RNA, DNA, or modified nucleic acids that bind with high affinity and specificity to various molecular targets, including proteins and peptides, has been described.
7 In particular, an RNA-based aptamer has been developed with high affinity toward VEGF
165.
8 After being isolated and determined to bind specifically to VEGF
165, RNA aptamer EYE001 (formerly referred to as NX1838) was further modified chemically to render it nuclease-resistant and thermally more stable, thus enhancing its potential for therapeutic utility.
7 8 The promising results displayed by the aptamer’s biological response both in vitro and in vivo against diseases associated with the growth of new blood vessels or angiogenesis, especially those threatening to vision, suggest that it has excellent potential as a therapeutic agent.
7 8 9 Currently, EYE001 is undergoing clinical trials for the treatment of age-related macular degeneration (AMD). Because choroidal neovascularization is a severe complication of AMD and because patients with subretinal neovascularization, including those with AMD, show increased expression of VEGF, it is believed that antiangiogenic and/or antivascular permeability factors could delay or reverse the pathogenesis of AMD.
10 11 12
Widespread clinical use of EYE001 will necessitate a practical and effective method of delivery to the eye. Application in current clinical trials relies on intravitreal injections of the aptamer. Although this method allows for assessment of the potential use of the aptamer as a therapeutic drug, it is a less than optimal way to treat patients on a day-to-day basis, because of its invasive nature. The mode of delivery should provide exposure to the drug for the required period it must be minimally invasive and, preferably, localized.
Recent studies have highlighted the applicability of transscleral delivery for various macromolecules, including globular proteins.
13 14 The potential for transport or diffusion through the sclera lies in the large and accessible surface area of this tissue, its high degree of hydration, hypocellularity, and permeability that does not decline significantly with age.
15 16 17 Thus, this approach would circumvent the limitations and problems presented by other modes of delivery to treat posterior segment diseases—intravitreal, systemic, and eye drops—that include, among other drawbacks, retinal detachment, systemic side effects, and diffusional limitations, respectively.
18 19 20
Because certain methods of transscleral delivery can be destructive (i.e., iontophoresis), causing, in some cases, retinal necrosis and gliosis,
21 we have focused our attention on biodegradable polymer sustained–delivery devices. Sustained delivery of proteins or nucleic acids from polymer matrixes offers the advantage of targeting specific tissues and increasing the comfort and compliance of patients.
22 23 24 Specifically, we have used poly(lactic-
co-glycolic) acid (PLGA) as the encapsulation matrix of choice. PLGA, an FDA-approved material, has been extensively studied for its biocompatibility, toxicology, and degradation kinetics.
25 26 It has been used clinically as a suture material since the 1970s,
27 and recently it has been used as scaffold in tissue engineering techniques.
28 29 An important characteristic of PLGA carrier systems is their ability to be applied locally, which allows intralesional concentrations of the drug to be sustained while systemic deleterious side effects are minimized, thus providing a pharmacological advantage at the treatment site.
30 31 In vivo studies in which PLGA was used as a carrier system to the eye for various types of drugs used to treat various diseases have reported no sign of ocular toxicity or significant inflammatory responses for periods of up to 2 months.
31 32 These studies, however, involved either the encapsulation of small synthetic drugs and molecules or the intravitreal injection of such polymer devices after a sclerotomy, which in itself is invasive.
In our present study, we considered the potential of transscleral delivery of drugs in a sustained and controlled manner in an in vitro setup. We were able to deliver the anti-VEGF aptamer EYE001 for a period of up to 20 days in a biologically active state, showing no destabilization due to the encapsulation procedure. We hypothesize that delivering drugs in a sustained manner through the sclera is a viable approach for the treatment of various vision-threatening diseases.
Human umbilical vein endothelial cells (HUVECs) were obtained from Cascade Biologics, Inc. (Portland, OR). Cells were maintained in growth-factor supplemented medium, including 2% vol/vol fetal bovine serum (FBS), 1 μg/mL hydrocortisone, 10 ng/mL human epidermal growth factor, 3 ng/mL basic fibroblast growth factor, and 10 μg/mL heparin under standard tissue culture conditions (5% CO2, 37°C, 100% relative humidity). Medium was changed every 48 to 72 hours, and cells were passaged by standard trypsinization and plated at a cell concentration of 2.5 × 103 cells/cm2.
Tissue was placed in modified Karnovsky fixative consisting of 2.5% glutaraldehyde and 2% formaldehyde in 0.1 M cacodylate buffer with 8 mM CaCl2 and fixed for 12 to 24 hours at 4°C. The specimens were subsequently changed to 0.1 M cacodylate buffer for storage at 4°C. The tissue was trimmed to block size and postfixed in 2% aqueous OsO4 for 2 hours at room temperature. After the tissue was rinsed in buffer, it was dehydrated in ascending concentrations of ethanol, transitioned through propylene oxide, and infiltrated with mixtures of propylene oxide and Epon (EMBed 812; Electron Microscopy Sciences, Fort Washington, PA), embedded in pure Epon, and polymerized at 60°C for 18 to 24 hours. One-micrometer sections and thin sections were cut on an ultramicrotome (Ultracut E; Leica, Deerfield, IL). The 1-μm sections were stained with 0.5% toluidine blue and the thin sections with saturated aqueous uranyl acetate and Sato lead stain, and then examined with a transmission electron microscope (model CM-10 Philips, Eindhoven, The Netherlands).
The goal of the present study was to develop a drug delivery modality that could release the anti-VEGF aptamer EYE001 in a sustained and controlled manner over a significant period and could be applied locally to the outer part of the sclera. The retina and choroid are the target tissues, because this aptamer is intended to block the contribution of VEGF to choroidal neovascularization and diabetic macular edema, respectively. Transscleral administration, no more frequently than every 6 weeks, would prove an attractive substitute to intravitreal injections of the aptamer, currently occurring at a similar frequency in two separate clinical trials.
For this purpose, the biodegradable, biocompatible, and FDA-approved polymeric material PLGA was selected.
23 The release profiles of EYE001 from these microspheres were characterized by a low initial burst, followed by continuous release in the absence of a lag phase. Typical release profiles from PLGA microspheres are triphasic, characterized by an initial burst as drug entrapped near the surface releases, followed by a lag phase controlled by polymer degradation and final release of the drug as it diffuses from the polymer core as erosion takes place.
26 In the scenario observed, it is probable that EYE001 formulations encapsulated in PLGA were homogeneously distributed throughout the polymeric matrix. As described by Higuchi
41 42 and reported elsewhere,
43 44 when a drug releases from a homogeneous matrix-type delivery system, the process is diffusion-controlled and is evidenced by a proportionality between the amount of drug being released and the square root of time. The process is described by the following equation:
\[Q{=}\sqrt{2WDC_{\mathrm{s}}t}\]
where
Q is the rate of released drug,
D is the diffusion coefficient of the drug in the matrix,
W is the total amount of the drug per unit volume of matrix,
C s is the solubility of the drug in the matrix, and
t is the drug release time.
The release of both excipient-free aptamer and EYE001-Tre from PLGA as a function of the square root of time (
t 1/2) show a linear relationship with correlation coefficients of 0.98 and 0.99, respectively
(Fig. 3B) . These data support the hypothesis that both aptamer formulations were released through a diffusion-controlled process.
An important consideration in our development of a long-term delivery device for a nucleic acid such as EYE001 was its stability before, during, and after the encapsulation process in PLGA. Nucleic acids are known to suffer depurination and become susceptible to free radical oxidation in aqueous solutions.
45 46 Such a phenomenon was recently reported by a group evaluating the potential development of pharmaceutical formulations of plasmid DNA with long-term storage stability.
47 To this end, we colyophilized EYE001 with the known potent stabilizer trehalose,
34 48 and used a completely nonaqueous oil-in-oil methodology
48 49 for the creation of polymer microspheres that has been effective in the delivery of biologically active proteins with native secondary structures.
24 48 49 50 51
The cell proliferation assays conducted to monitor aptamer bioactivity after release from PLGA microspheres reveal that the conditions chosen to create the polymer microspheres were satisfactory. As shown in
Figure 5 , EYE001 preserved its ability to inhibit VEGF-induced cell proliferation during all the representative time points along its release from PLGA. Although bioactivity was retained regardless of its formulation state, an improved level of bioactivity was observed in general when EYE001 was colyophilized with trehalose before encapsulation.
Incubation of PLGA microspheres directly with HUVECs revealed the same trend as that of the aptamer collected after it was released from isolated microspheres in vitro. No evident signs of toxicity or cell death were observed when blank PLGA microspheres were incubated with HUVECs from microscopic observations and cell counts (data not shown). These results are in agreement with reports by other groups that conducted cell proliferation assays with polylactides of various molecular weights with rat epithelial cells, human fibroblasts, and osteosarcoma cells under culture conditions.
52 Overall, it was determined that satisfactory biocompatibility was exhibited.
52 53 These data add support for the conclusion that the method described in this report holds promise for the long-term inhibition of VEGF-mediated responses in vivo.
Rabbit sclera is 71% water
17 and, as documented by electron microscopy
(Fig. 6C) , it served to hydrate and degrade the solid PLGA microspheres placed on the orbital side of the sclera, which were not in contact with any hydration medium other than the hydrated scleral surface itself. An important aspect of PLGA controlled-delivery devices is that they provide continuous release and avoid the repeated use of injections or high concentrations of drug to achieve the desired pharmacological response. Even though controversy exists over how the flux over the sclera occurs and whether it achieves steady state,
54 our controlled-drug delivery device would increase drug–sclera contact, thus improving scleral absorption. The hypocellularity
55 and large surface area
15 of the human sclera, as well as its remarkable tolerance of foreign bodies overlying its surface (e.g., scleral buckles
56 ) helps to facilitate diffusion through it and allow a long-term transscleral delivery device to be clinically feasible.
In this report, we present data showing the feasibility of delivering the anti-VEGF aptamer EYE001 in a sustained and controlled manner and in a biologically active form. The development of such an approach to drug delivery accompanies the advent of many potential antiangiogenic drugs for the treatment of various vision-threatening diseases that affect the posterior segment of the eye. Validation of this study would require testing the system in an in vivo model that would also address other important questions: how choroidal blood flow affects transscleral delivery and whether the concentrations of active drug delivered through the proposed system are sufficient to inhibit some or all the responses triggered by neovascularization in the posterior segment, among others. These studies are currently in progress in our laboratory.
Supported by Eyetech Pharmaceuticals (APA), the Roberta W. Siegel Fund (APA), National Eye Institute Grants EY12611 and EY11627 (APA), the Juvenile Diabetes Foundation (APA), the Falk Foundation (APA), and the Iaccoca Foundation (APA).
Submitted for publication November 19, 2001; revised May 17, 2002; accepted June 18, 2002.
Commercial relationships policy: F (KGC, IKR, APA); P (JWM, ESG, APA); C (APA); N (JAR).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “
advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Anthony P. Adamis, Massachusetts Eye and Ear Infirmary, 325 Cambridge Street, Boston, MA 02114;
[email protected].
Table 1. Amount of Aptamer Diffused through the Sclera after Release from PLGA Microspheres
Table 1. Amount of Aptamer Diffused through the Sclera after Release from PLGA Microspheres
Day | EYE001SC * (μg) | EYE001CC , † (μg) |
1 | 3.4 ± 0.8 | 0.7 ± 0.3 |
2 | 2.3 ± 0.5 | 0.5 ± 0.3 |
3 | 2.1 ± 0.6 | 0.5 ± 0.2 |
4 | 1.8 ± 0.3 | 0.6 ± 0.4 |
5 | 2.4 ± 0.1 | 0.5 ± 0.2 |
6 | 2.6 ± 0.2 | 0.4 ± 0.3 |
The authors thank Norman Michaud, Director of Morphology, from the Howe Laboratory, Division of Ophthalmology of the Massachusetts Eye and Ear Infirmary for assistance with the transmission electron microscopic studies, Jeffrey T. Borenstein, Draper Laboratories, for assistance with the scanning electron microscopy, and Carlos J. Bosques, Department of Chemistry, Massachusetts Institute of Technology, for assistance with the circular dichroism studies.
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