NIH Public Access
`Author Manuscript
`Exp Eye Res. Author manuscript; available in PMC 2014 November 01.
`Published in final edited form as:
`Exp Eye Res. 2013 November ; 116: . doi:10.1016/j.exer.2013.09.001.
`
`Ocular silicon distribution and clearance following intravitreal
`injection of porous silicon microparticles
`
`Alejandra Nietoa,b,1, Huiyuan Houa,b,1, Michael J. Sailorb, William R. Freemana, and Lingyun
`Chenga,*
`Lingyun Cheng: cheng@eyecenter.ucsd.edu
`aJacobs Retina Center at Shiley Eye Center at University of California, San Diego, USA
`bDepartment of Chemistry and Biochemistry, University of California, San Diego, USA
`
`Abstract
`Porous silicon (pSi) microparticles have been investigated for intravitreal drug delivery and
`demonstrated good biocompatibility. With the appropriate surface chemistry, pSi can reside in
`vitreous for months or longer. However, ocular distribution and clearance pathway of its
`degradation product, silicic acid, are not well understood. In the current study, rabbit ocular tissue
`was collected at different time point following fresh pSi (day 1, 5, 9, 16, and 21) or oxidized pSi
`(day 3, 7, 14, 21, and 35) intravitreal injection. In addition, dual-probe simultaneous microdialysis
`of aqueous and vitreous humor was performed following a bolus intravitreal injection of 0.25 mL
`silicic acid (150 μg/mL) and six consecutive microdialysates were collected every 20 min. Silicon
`was quantified from the samples using inductively coupled plasma-optical emission spectroscopy.
`The study showed that following the intravitreal injection of oxidized pSi, free silicon was
`consistently higher in the aqueous than in the retina (8.1 ± 6.5 vs. 3.4 ± 3.9 μg/mL, p = 0.0031).
`The area under the concentration-time curve (AUC) of the retina was only about 24% that of the
`aqueous. The mean residence time was 16 days for aqueous, 13 days for vitreous, 6 days for
`retina, and 18 days for plasma. Similarly, following intravitreal fresh pSi, free silicon was also
`found higher in aqueous than in retina (7 ± 4.7 vs. 3.4 ± 4.1 μg/mL, p = 0.014). The AUC for the
`retina was about 50% of the AUC for the aqueous. The microdialysis revealed the terminal half-
`life of free silicon in the aqueous was 30 min and 92 min in the vitreous; the AUC for aqueous
`accounted for 38% of the AUC for vitreous. Our studies indicate that aqueous humor is a
`significant pathway for silicon egress from the eye following intravitreal injection of pSi crystals.
`
`Keywords
`intravitreal porous silicon; ocular silicon clearance; ocular drug delivery; rabbit eye
`
`1. Introduction
`Retinal diseases are often chronic and refractory to treatment. Two unique challenges in
`developing drugs against these diseases derive from the inherent barriers that prevent drugs
`in the blood from reaching the retina and from the short vitreous half-life of drugs
`
`© 2013 Elsevier Ltd. All rights reserved.
`*Corresponding author. Department of Ophthalmology, Jacobs Retina Center at Shiley Eye Center, University of California, San
`Diego, 9415 Campus Point Drive, La Jolla, CA 92093-0946, USA. Tel.: +1 858 534 3780; fax: +1 858 534 7985.
`1Both authors contributed equally to this work.
`Financial disclosure: A. Nieto, None; H. Hou, None; M.J. Sailor, Spinnaker Biosciences (I); W.R. Freeman, Spinnaker Biosciences
`(C); L. Cheng, Spinnaker Biosciences (C).
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`Exhibit 2062
`Page 01 of 22
`
`

`

`Nieto et al.
`
`Page 2
`
`administered via intravitreal injection. This makes necessary frequent drug administration if
`directly injected into the eye, entailing the risk of infection and other complications. A major
`focus in our research is to devise strategies for bypassing this barrier and developing ocular
`drug delivery systems able to provide sustained drug levels to the retina (Cheng et al., 2004;
`Chhablani et al., 2013).
`
`Mesoporous silicon microparticles have been proposed as a drug delivery vehicle to achieve
`a slow drug release and a long-lasting therapeutic effect. (Anglin et al., 2008; Salonen et al.,
`2008), (Kashanian et al., 2010) In general, porous silicon has demonstrated good
`biocompatibility. (Bimbo et al., 2010) However, biocompatibility is dependent on location
`and tissue types (Leoni et al., 2002; Park et al., 2009) as well as surface chemistry of the
`porous silicon particles. (Low et al., 2006) The eye is a unique organ with clear media that
`offers a direct view of compounds or delivery systems injected into the vitreous. The eye is
`especially suitable for a porous silicon drug delivery system because the direct imaging of
`porous silicon particles may enable non-invasive monitoring of payload release from the
`nanoscale pores. (Wu et al., 2011) We previously showed that intravitreally injected porous
`silicon particles were not toxic in rabbit eyes, showing good biocompatibility and slow
`elimination from the body. (Cheng et al., 2008) Elimination of micron-size particles of
`porous silicon from the eye necessarily involves degradation of the material into water-
`soluble products. The degradation of porous silicon in vivo involves two main processes:
`oxidation of the elemental silicon component and dissolution of the silicon oxide thus
`formed. The soluble silicon-containing species are various protonated forms of the
`, and its oligomers. The form of orthosilicate that predominates at
`orthosilicate ion,
`neutral pH is silicic acid Si(OH)4, which is naturally found in numerous tissues. Silicon is
`considered an essential trace element and in aqueous environment at neutral pH, Si(OH)4 is
`excreted from the body through the urine (Mertz, 1981). For the purpose of ocular drug
`delivery, a significant quantity of porous silicon particles needs to be placed in the vitreous.
`It is therefore important to assess the fate of the silicic acid degradation product of the
`particles. The ocular distribution and clearance pathway of silicic acid has not yet been
`documented. This issue is not only of academic interest, but it is also a practical issue to
`better understand this ocular drug delivery system and possible safety issues associated with
`higher doses and longer-term exposure of ocular tissues involved in the clearance pathway
`of silicic acid. Theoretically, porous silicon particles degrade in the vitreous and the final
`product, silicic acid, may be removed from the eye through the retina to the choroid then to
`systemic circulation or through the aqueous humor to Schlemm's canal and then to systemic
`circulation. The surface chemistry plays an important role in the rate at which particles
`degrade; as-prepared porous silicon degrades faster than material whose surface has been
`modified by oxidization or hydrosilylation (Cheng et al., 2008). The purpose of the present
`study was to determine the ocular elimination kinetics and pathways of silicon clearance
`from the eye for two important formulations of porous silicon microparticles: as-prepared
`particles (pSi) which consist of Si–Si and Si–H bonds, and thermally oxidized porous silicon
`particles (pSiO2) which contain Si–O–Si bonds as the primary structural motif. The
`correlation of pSi degradation with drug release will depend on individual drug loaded such
`as daunorubicin shown previously by us (Wu et al., 2011). However, the silicon clearance
`pathway is independent to payload and is the objective of the current study.
`2. Materials and methods
`Porous silicon (pSi) microparticle preparation
`Porous Si (pSi) microparticles were prepared by anodic electrochemical etch of highly
`doped, (100)-oriented, p-type silicon wafers (boron-doped, 1.10 mΩ·cm resistivity; obtained
`from Siltronix Inc., Archamps, France), in an electrolyte consisting of a 3:1 (v:v) solution of
`
`Exp Eye Res. Author manuscript; available in PMC 2014 November 01.
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`Exhibit 2062
`Page 02 of 22
`
`

`

`Nieto et al.
`
`Page 3
`
`48% aqueous hydrofluoric acid (HF) and ethanol (Fisher-Scientific, Pittsburg, PA). A Si
`wafer with an exposed area of 8.04 cm2 was contacted on the back side with a strip of
`aluminum foil and mounted in a Teflon etching cell that was fitted with a platinum counter
`electrode. The wafer was etched using a current density waveform (J) previously described:
`(Wu et al., 2011) J = A0 +A·cos(kt+ α), where A0 is current density offset (mA/cm2), A is
`current density amplitude (mA/cm2), k is frequency (s−1), t is time (s), and a is phase shift
`(s−1). The values used for A0, A, and α were 90.2 mA/cm2,12.4 mA/cm2, and 0, respectively.
`The current density waveform generates a porosity modulation in the porous silicon layer
`that acts as a 1-dimensional photonic crystal. (Vincent, 1994), (Thonissen and Berger, 1997)
`The photonic crystal displays a sharp peak in the optical reflectance spectrum whose
`wavelength is directly proportional to k. The value of k was adjusted to yield a reflectance
`peak whose maximum occurred in the spectrum at ∼600 nm; a typical value for k was 2.25.
`The waveform was etched into the silicon wafer for a total of 400 s. The resulting porous
`layer was then removed from the silicon substrate by replacing the electrolyte with a 1:29
`(v:v) solution of 48% aqueous hydrofluoric acid and ethanol and then applying an anodic
`pulse (6.2 mA/cm2) for 120 s. Each wafer was subjected to four such etching protocols, and
`the porous layers were collected and ultrasonicated in ethanol for 30 min in an FS5 dual
`action ultrasonic cleaner (Thermo Fisher Scientific, Pittsburg, PA), then rinsed with ethanol
`3 times.
`
`Porous silicon oxide (pSiO2) microparticle preparation
`The pSiO2 formulation was prepared from pSi by air oxidation in a ceramic boat inside a
`muffle furnace (Thermo Fisher Scientific, Pittsburg, PA). The temperature in the furnace
`was increased from room temperature to 800 °C at a heating rate of 10 °C min−1 and then
`held in air for 1 h. The furnace was allowed to cool to room temperature for an additional 3
`h prior to removal of the samples. The oxidation reaction was accompanied by a color
`change of the particles from brown to transparent, corresponding to conversion of Si to SiO2
`in the porous matrix. However, the particle size and shape were not affected by the oxidation
`procedure. For sustained ocular drug delivery purposes, we have found that oxidized porous
`silicon shows a longer vitreous half-life (Cheng et al., 2008) and the surface is more
`amenable to covalent functionalization for subsequent drug grafting (Chhablani et al., 2013).
`
`Physical characterization of porous microparticles
`Average particle size and pore size were determined from plan-view images of randomly
`selected particles (n > 10) using a Phillips XL30 field emission scanning electron
`microscope operating at an accelerating voltage of 5 kV (FEI Phillips, Hillsboro, OR).
`Surface chemistry was characterized by Fourier transform infrared (FTIR) spectroscopy
`using attenuated total reflectance (ATR) mode on a Nicolet 6700 Smart-iTR spectrometer
`(Thermo Fisher Scientific, Pittsburg, PA). The textural properties of the particles were
`analyzed by nitrogen adsorption at −196 °C on an ASAP 2020 porosimetry apparatus
`(Micromeritics, Norcross, GA). Prior to the adsorption experiment, approximately 50 mg of
`the porous Si sample was outgassed overnight at 105 °C. The specific surface area and pore
`volume of the particles were calculated from the N2 adsorption isotherms using the BET
`(Brunauer-Emmett-Teller) and BJH (Barrett-Joyner-Halenda) methods, respectively (Gregg
`and Sing, 1982), (Brunauer et al., 1938; M. Kruk and Jaroniec, 1999).
`
`Animal studies
`All animal experiments were carried out in adherence to ARVO Statement for the Use of
`Animals in Ophthalmic and Vision Research.
`
`1) Ocular pharmacokinetic tissue sampling: Forty pigmented rabbits were used for the study
`and only one eye of each rabbit was used for microparticle intravitreal injection. Out of 40
`
`Exp Eye Res. Author manuscript; available in PMC 2014 November 01.
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`Exhibit 2062
`Page 03 of 22
`
`

`

`Nieto et al.
`
`Page 4
`
`rabbits, 20 were used for the fresh porous silicon (pSi) particle study and the other 20 were
`used for the oxidized porous silicon (pSiO2) particle study. For the pSi particle study, 1 mg
`of pSi particles in 100 μL of balanced salt solution (BSS; Thermo Fisher Scientific,
`Pittsburg, PA) was injected into the right eye of each of the 20 rabbits using a 27 gage
`needle. At the post-injection time points day 1, 5, 9, 16, and 21, four rabbits were subjected
`to a comprehensive eye exam, including anterior segment biomicroscopy, posterior indirect
`ophthalmoscopy and intraocular pressure (IOP) measurements, before the planned sacrifice.
`Four animals at each time point were used to account for the inherent variation of pSi
`microparticle suspension concentration injected as well as variation between individual
`animals. Immediately after sacrifice, the enucleated eye globes were dissected individually
`into aqueous, vitreous, and retina as described previously (Cheng et al., 2004; Nan et al.,
`2010). For the pSiO2 particle study, 2 mg of particles in 100 μL of BSS were injected with a
`27 gage needle and a longer study course was performed because of the slower dissolution
`property of pSiO2 microparticles (Cheng et al., 2008). The rabbits injected with the pSiO2
`particles were sacrificed at post-injection day 3, 7, 14, 21, and 35, four animals at each time
`point. Prior to both intravitreal injection and the scheduled sacrifice, 1 mL of blood was
`sampled for silicon quantitation. The collected eye tissues and plasma were kept at −80 °C
`until the quantitation of Si (as dissolved silicic acid) was performed by inductively coupled
`plasma-optical emission spectroscopy (ICP-OES).
`
`2) Simultaneous microdialysis of aqueous and vitreous humor: To better understand the
`distribution and elimination of orthosilicate from the eye, a dual-probe microdialysis was
`performed as previously described (Chen et al., 2013). For this acute study, one rabbit was
`used (3.7 kg body weight). The rabbit had a comprehensive eye exam before the
`microdialysis experiment. After general anesthesia with intramuscular ketamine (35 mg/kg
`body weight) and xylazine (6.25 mg/kg body weight) and topical anesthesia with
`proparacaine hydrochloride (Ophthalmic solution USP 0.5% (wt/vol); Bausch & Lomb,
`Rochester, NY), an anterior chamber microdialysis probe (CMA 30 linear, 4 mm membrane
`length custom made, 6000 Da molecular weight cut-off; CMA Microdialysis, North
`Chelmsford, MA) was installed followed by installation of a vitreous microdialysis probe
`(CMA 20 Elite, 4 mm membrane length, 20,000 Da molecular weight cut-off; CMA
`Microdialysis, North Chelmsford, MA). Tissue adhesive (Vetbond 1469SB; 3M
`Corporation, St Paul, MN) was applied around the probe entry point to prevent ocular fluid
`leaks. Thirty minutes after the installation of the probes, intravitreal injection of 0.250 mL of
`silicic acid solution (150 μg/mL) was performed to yield final vitreous concentration of 25
`μg/mL, assuming rabbit vitreous volume to be 1.5 mL The saturated stock silicic acid
`solution was prepared by dissolving 5 mg pure silicic acid crystals (Spectrum Laboratory
`Products, Gardena, CA) in 10 mL 50 mM sodium hydroxide (NaOH; Sigma–Aldrich, St
`Louis, MO) in phosphate buffered saline solution (PBS; Thermo Fisher Scientific, Pittsburg,
`PA) and incubated at 37 °C for one week. The solution pH was adjusted to 7 with
`hydrochloric acid (HCl; Sigma–Aldrich, St Louis, MO) before intravitreal injection of the
`supernatant. During the microdialysis experiment, both probes were perfused with PBS at 1
`μL/min using a microsyringe pump (NE-100; New Era Pump Systems Inc, Farmingdale,
`NY). The vitreous and aqueous perfusate samples were collected every 20 min for total of 6
`collections. During the course of the microdialysis experiment, the rabbit remained on a
`water blanket (TP650; Gaymar Industries, Orchard Park, NY) to help prevent body heat loss
`and anesthesia was maintained by boosting every 40 min using one half volume of the first
`dose. Every other dose was ketamine only starting with the first boost because xylazine stays
`in the system longer (Veilleux-Lemieux et al., 2012). At the end of the microdialysis
`procedure, a blood sample was acquired before the animal was sacrificed and the eye globe
`enucleated. The eye globe was dissected under a surgical microscope to sample aqueous
`humor, vitreous humor, and retina. These eye tissues were separately stored at −80 °C until
`silicon was quantitated using ICP-OES.
`
`Exp Eye Res. Author manuscript; available in PMC 2014 November 01.
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`Exhibit 2062
`Page 04 of 22
`
`

`

`Nieto et al.
`
`Page 5
`
`After the in vivo microdialysis experiment, the probes were placed in deionized water (WFI-
`quality, cell culture grade, Cellgro, Manassas, VA) for continued flushing overnight to clean
`the silicic acid from the system before performing a test to determine the recovery rate of
`silicon for the probes. For this purpose, each probe was soaked in a vial of 25 (μg/mL silicic
`acid solution and the perfusate was collected every 20 min for 6 consecutive times.
`
`Silicon quantification
`To determine the silicon content as dissolved silicon (silicic acid) in the aqueous and
`vitreous, the collected fluid samples were weighed and centrifuged at 11,000 rpm for 10
`min. Aqueous fluid supernatant (0.1 mL) was diluted with 2% (v/v) nitric acid aqueous
`solution to make a final volume of 3 mL (HNO3; EMD, Darmstad, Germany). Vitreous fluid
`supernatant (0.07 mL) was diluted with 2% (v/v) HNO3 aqueous solution to make a final
`volume of 3 mL (Park et al., 2009), (Dyck et al., 2000; Hauptkorn et al., 2001; Wills et al.,
`2008) To determine the silicon content in the retina tissue, the wet samples were weighed
`and digested with hydrogen peroxide 30% (w/w) (0.05 mL) (H2O2; Sigma–Aldrich, St.
`Louis, MO) and concentrated HNO3 ∼ 15.7 M (0.250 mL) for 2 days. To determine the
`silicon concentration in plasma, 0.150 mL of sample was digested with a solution consisting
`of 0.025 mL of 30% (by mass) aqueous H2O2 and 0.125 mL of concentrated HNO3 for 2
`days. Mechanical homogenization of the tissue samples was performed with a pellet pestle
`(Sigma–Aldrich, St. Louis, MO) applied for 10 s after addition of the digesting reagents,
`resulting in a homogeneous slurry. The digested tissues were centrifuged at 11,000 rpm for
`10 min and the supernatant (0.1 mL) was diluted with 2% (v/v) HNO3 aqueous solution to
`make a final volume of 3 mL This open vessel digestion procedure with HNO3–H2O2 was
`designed to ensure that as much of the analyte that is available for recovery is rendered
`soluble and relatively stable in aqueous acidic medium. Silicon calibration standard
`solutions were prepared from a stock solution containing 1000 μg/mL Si (Trace Cert Silicon
`standard for ICP in nitric acid; Fluka, Milwaukee, WI). External calibration standard
`solutions were prepared in 2% (v/v) HNO3 solution in water (WFI-quality, cell culture
`grade; Cellgro, Manassas, VA) by serial dilution to reach final concentrations 8, 4, 2,1, 0.5,
`0.1 and 0.01 μg/mL Calibration standards and samples were prepared in polystyrene tubes.
`Normal rabbit aqueous, vitreous, retina and plasma from rabbits that had not received any
`injection of pSi or pSiO2 particles (InVision BioResources, Seattle, WA) were treated
`following the same procedure as the samples and used as controls to determine the baseline
`levels of silicon.
`
`Soluble silicon, in the form of protonated orthosilicate species, was detected by inductively
`coupled plasma-optical emission spectroscopy (ICP-OES) in an argon plasma spectrometer
`(Optima 3000 DV; Perkin–Elmer, Norwalk, CT) equipped with a standard torch, Scott-type
`spray chamber, GemTip cross-flow nebulizer and an AS-90 autosampler (Perkin–Elmer,
`Norwalk, CT). The ICP-OES instrument parameters are summarized in Table 1.
`
`Data analysis
`Pharmacokinetic (PK) parameters of silicon content in ocular tissues and plasma were
`calculated using non-compartmental methods and sparse sampling setting within the
`Phoenix® WinNonlin® software (version 6.3; Pharsight Corp, Mountain View, CA). The
`area under the curve from time zero to the last measurable concentration (AUC0–t) was
`calculated. A terminal rate constant of elimination was calculated using a minimum of three
`measurable concentrations and a terminal elimination half-life was calculated using 0.693/
`kel. The pharmacokinetic parameters of silicon content from the microdialysis of vitreous
`and aqueous humor were calculated using the PK software module using a one-compartment
`model and IV-Bolus input setting. For comparison of silicon concentrations detected among
`the ocular tissues, the data from all the time points of the same tissue were pooled and
`
`Exp Eye Res. Author manuscript; available in PMC 2014 November 01.
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`Exhibit 2062
`Page 05 of 22
`
`

`

`Nieto et al.
`
`Page 6
`
`nonparametric multiple comparison (each pair using Wilcoxon method) was performed
`using JMP statistical software (version 10; SAS Institute Inc, Cary, NC).
`
`3. Results
`Porous silicon particle characterization
`The pSi and pSiO2 particles displayed an average particle size of 43 × 70 × 25 μm and an
`average pore size of ∼13 nm in the electron microscope images (Fig. 1). Particle size and
`shape were not affected by the oxidation procedure.
`
`The FTIR spectrum of pSi microparticles display bands characteristic of surface hydride
`species. A band at 2110 cm−1 associated with the νSi-H stretching vibrations, a band
`corresponding to the δ Si-H2 scissors mode at 910 cm−1, and a band corresponding to a νSi-
`H deformation mode (665 cm−1) are apparent in the spectrum (Fig. 2, line tracing at the
`bottom). For the pSiO2 formulation, complete oxidation of the porous Si matrix to porous
`SiO2 was verified ex situ by the appearance of a broad band centered at 1000 cm−1 in the
`FTIR spectrum, associated with silicon-oxide stretching modes (Fig. 2, line tracing at the
`top).
`
`The N2 adsorption/desorption isotherms of the pSi and pSiO2 microparticles are plotted in
`Fig. 3. The isotherms exhibit type IV hysteresis loops with parallel adsorption and
`desorption branches suggesting cylindrical mesopores of approximately constant cross
`section. The measured surface area decreases after oxidation, from 454 ± 5 m2/g for pSi to
`207 ± 4 m2/g for pSiO2 microparticles. The total pore volume decreases from 1.3 cm3/g for
`pSi to 0.6 cm3/g for pSiO2.
`
`Clinical Exams
`The eyes injected with the oxidized particles did not show any abnormalities at any time
`points following the injection. Two eyes out of the twenty eyes injected with fresh pSi
`particles were found with mild vitreous cloudiness and aqueous trace cells (4–5 cells per
`millimeter square) on their scheduled sacrifice days (day 9 and day 16). The rabbit eyes at
`the other time points were normal. The intraocular pressure (IOP) was comparable between
`the injected eyes and their fellow eyes (for pSi particles, OD IOP = 9.4 ± 2.0 vs OS IOP =
`10.2 ± 2.6 mmHg, p = 0.46, t Test; for pSiO2 particles, OD IOP = 7.5 ± 1.7 vs OS IOP = 8.2
`± 1.3 mmHg, p = 0.27, t Test).
`
`Ocular Pharmacokinetics
`Normal silicon concentration value in eye and plasma—The silicon concentration
`found in the normal rabbit aqueous humor, vitreous humor, retina, and plasma is
`summarized in Table 2.
`
`Porous silicon particles (pSi)—The elimination of orthosilicate from the eye following
`a single intravitreal injection of 1 mg pSi particles is demonstrated in Fig. 4.
`
`The ocular pharmacokinetics of Si reflects both the degradation of pSi crystals to generate
`aqueous silicic acid and the clearance of this soluble aqueous silicic acid product. As shown
`in Fig. 4, detected free silicon (in the form of aqueous silicic acid) was consistently higher in
`the aqueous humor (mean 7 ±4.7 μg/mL) than in the retina (mean 3.4 ± 4.1 μg/mL) (p =
`0.014, each pair usingWilcoxon method), which suggests the aqueous humor is a significant
`pathway for silicon egress from the eye following an intravitreal injection. Accordingly, the
`area under the concentration-time curve of the retina was only about 50% of that of the
`aqueous humor (Table 3).
`
`Exp Eye Res. Author manuscript; available in PMC 2014 November 01.
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`Exhibit 2062
`Page 06 of 22
`
`

`

`Nieto et al.
`
`Page 7
`
`Free silicic acid concentration in the aqueous humor, the vitreous humor, and the retina was
`highest at the first time point (day 1) after the injection and gradually decreased over time.
`The mean residence time was 7 days for vitreous humor, 9 days for aqueous humor, 7 days
`for retina, and 11 days for plasma.
`
`Oxidized porous silicon particles (pSiO2)—The concentration-time traces for
`elimination of silicic acid from the eye following a single intravitreal injection of 2 mg of
`pSiO2 particles is shown in Fig. 5.
`
`Similar to the elimination data observed following intravitreal injection of pSi, the ocular
`elimination pharmacokinetics of pSiO2 represents both the dissolution kinetics of the porous
`silicon matrix and the clearance of the soluble silicon product, silicic acid. As expected, the
`concentration of free silicic acid was the largest in the vitreous humor, and the concentration
`of free silicic acid was significantly larger in the aqueous humor than in the retina (vitreous
`mean 15 ± 9 vs aqueous mean 8.1 ± 6.5, p = 0.0063; aqueous mean vs retina mean of 3.4 ±
`3.9, p = 0.0031; each pair using Wilcoxon method). The area under the concentration-time
`curve for the retina was only about 24% of the area under the corresponding curve for the
`aqueous humor (Table 4). This distribution of soluble silicon among the three ocular tissues
`was similar to the clearance profile observed for the pSi particle formulation.
`
`Microdialysis of aqueous and vitreous humor—In experiments involving dual-probe
`microdialysis of vitreous and aqueous humor, the silicon was administered in its free soluble
`form (silicic acid) as a bolus injection into the vitreous. The recovery rate for the probe in
`the vitreous humor was 13% and in the aqueous humor it was only 2%. The kinetics of
`elimination of silicic acid from vitreous and from aqueous humor both followed a first-order
`one-compartment PK model (Fig. 6).
`
`The terminal half-life of soluble silicon in the aqueous humor was 30 min and in the vitreous
`humor it was 92 min; the area under the concentration-time curve for aqueous humor was
`1490 which was 38% of the corresponding area under the curve for vitreous humor (3950)
`(Table 5).
`4. Discussion
`The current study showed that elevated silicon levels were detectable in the aqueous, the
`vitreous, and the retina of the rabbit eyes as well as in the animal's systemic circulation
`following a single intravitreal injection of the pSi or pSiO2 particles. It appears that the
`silicic acid dissolution product of the pSi or pSiO2 particles in the vitreous humor migrated
`forward into the aqueous humor more effectively than backwards into the retina because the
`dissolved silicon levels were significantly larger in the aqueous humor than in the retina for
`both types of injected particles. The pharmacokinetic analysis demonstrated that the
`partitioning of silicic acid to the retina was only 24% (pSi particle study) to 50% (pSiO2
`particle study) of the quantity that migrated to the aqueous humor. This is in contradiction
`with the normal vitreous fluid elimination pathway. It is believed that vitreous water exits
`mainly through the retina into the choroid (over 80%) and only a very small percentage
`(<5%) is removed through the aqueous pathway (Moseley et al., 1984). The current results
`indicate that dissolved Si in the form of silicic acid may not passively diffuse along with
`vitreous water elimination. A similar situation has also been reported for some other small
`molecules by Araie and Maurice, who demonstrated that intravitreally injected fluorescein
`mainly leaves the vitreous compartment through the retinal surface while similarly injected
`fluorescein glucuronide and dextran-conjugated fluorescein isothiocyanate leaves the
`vitreous compartment through the vitreous–aqueous interface and the aqueous pathway
`(Araie and Maurice, 1991). The mechanism that favors elimination of silicic acid through
`
`Exp Eye Res. Author manuscript; available in PMC 2014 November 01.
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`Exhibit 2062
`Page 07 of 22
`
`

`

`Nieto et al.
`
`Page 8
`
`the anterior segment rather than the posterior segment is not understood at this time,
`although it may be related to the charge on the silicic acid species. While silicic acid
` at pH >
`(Si(OH)4) is a neutral species at pH 7, it is deprotonated to form the ion
`10. Silicic acid can also form complexes with metal cations, (Collery et al., 1998; Iler, 1979)
`and at high concentrations it can form larger molecular weight oligomers (Brinker and
`Scherer, 1990). We hypothesize that ions or complexes of mono-meric silicic acid (Si(OH)4)
`may form in the vitreous that are less capable of penetrating the retina. It should be noted
`that how the degraded product (silicic acid) of porous silicon is eliminated will not affect
`depository property of porous silicon as a drug delivery vehicle in vitreous. How effective
`the payload can target retina depends on the property of the payload once leached out from
`porous silicon into vitreous; and how long the crystal porous silicon can last in vitreous
`depends on its stability imparted by surface chemistry modifications.
`
`As mentioned above, the pharmacokinetics of pSi microparticles represents a combination
`of the kinetics of oxidation of silicon and dissolution of the oxide formed. The oxide on the
`pSi formulation is formed at physiological conditions, whereas and the oxide contained in
`the pSiO2 formulation is prepared at 800 °C. The chemical behavior of these two oxides is
`expected to be quite different. Indeed, in our previous study we found that as-formed porous
`silicon (pSi) degrades faster than oxidized porous silicon (pSiO2) when placed in rabbit
`vitreous (Cheng et al., 2008). The more rapid degradation of the pSi formulation is expected
`to yield a larger concentration of dissolved silicon in the vitreous fluid. To allow better
`comparison of the pSi and the pSiO2 results, we therefore injected a smaller quantity of pSi
`particles compared to the pSiO2 particles (1 mg versus 2 mg, respectively). In either case,
`the injected particles act as a depot that gradually degrades in the vitreous to yield a
`sustained concentration of dissolved silicon in all the ocular tissues studied. The non-
`compartmental pharmacokinetic analysis showed that both mean residence time (MRT) and
`terminal half-life of the pSiO2 particles were larger than those of the pSi particles, which is
`expected due to the slower rate of pSiO2 degradation in vivo. (Cheng et al., 2008) Though
`the elimination kinetics of Si in these two studies is different, in both cases the exposure of
`the aqueous humor to Si was significantly greater than the exposure of the retina to Si.
`
`To confirm the findings described above we injected dissolved silicic acid into the vitreous
`and performed simultaneous microdialysis from the vitreous and aqueous humor. The
`elimination kinetics of Si from both the vitreous and the aqueous fit well to a one-
`compartment PK model. Si elimination from the aqueous compartment was faster (25 ng/
`min) and the half-life was shorter (30 min) compared with the vitreous compartment (9 ng/
`min and half-life of 92 min, respectively). The results are consistent with fluid turnover rates
`for aqueous and vitreous of rabbit in that the turnover rate in the aqueous humor is ∼4 μL/
`min while it is only ∼2 μL/min in the vitreous (McMaster and Macri, 1967), (Becker, 1961)
`(Davson and Luck, 1956). As was observed with the particle injections, we found that
`significant quantities of silicon transfer to the aqueous compartment: integration of the
`curves of Fig. 6 showed that the concentration of Si in the aqueous compartment was 38% of
`the concentration in the vitreous compartment. It should be noted that the microdialysis was
`to confirm if a significant part of intravitreally injected soluble silicic acid is indeed
`eliminated from the anterior chamber pathway; the vitreous or aqueous half-life of the
`soluble silicic acid should not be confused with the half-life of the crystal porous silicon
`particle.
`
`In the current study, silicon levels measured in plasma were pers