Huiyuan Hou

Tunable sustained intravitreal drug delivery system for daunorubicin using oxidized porous silicon.

Daunorubicin (DNR) is an effective inhibitor of an array of proteins involved in neovascularization, including VEGF and PDGF. These growth factors are directly related to retina scar formation in many devastating retinal diseases. Due to the short vitreous half-life and narrow therapeutic window, ocular application of DNR is limited. It has been shown that a porous silicon (pSi) based delivery system can extend DNR vitreous residence from a few days to 3months. In this study we investigated the feasibility of altering the pore size of the silicon particles to regulate the payload release. Modulation of the etching parameters allowed control of the nano-pore size from 15nm to 95nm. In vitro studies showed that degradation of pSiO2 increased with increasing pore size and the degradation of pSiO2 was approximately constant for a given particle type. The degradation of pSiO2 with 43nm pores was significantly greater than the other two particles with smaller pores, judged by observed and normalized mean Si concentration of the dissolution samples (44.2±8.9 vs 25.7±5.6 or 21.2±4.2μg/mL, p<0.0001). In vitro dynamic DNR release revealed that pSiO2-CO2H:DNR (porous silicon dioxide with covalent loading of daunorubicin) with large pores (43nm) yielded a significantly higher DNR level than particles with 15 or 26nm pores (13.5±6.9ng/mL vs. 2.3±1.6ng/mL and 1.1±0.9ng/mL, p<0.0001). After two months of in vitro dynamic release, 54% of the pSiO2-CO2H:DNR particles still remained in the dissolution chamber by weight. In vivo drug release study demonstrated that free DNR in the vitreous at post-injection day 14 was 66.52ng/mL for 95nm pore size pSiO2-CO2H:DNR, 10.76ng/mL for 43nm pSiO2-CO2H:DNR, and only 1.05ng/mL for 15nm pSiO2-CO2H:DNR. Pore expansion from 15nm to 95nm led to a 63 fold increase of DNR release (p<0.0001) and a direct correlation between the pore size and the drug levels in the living eye vitreous was confirmed. The present study demonstrates the feasibility of regulating DNR release from pSiO2 covalently loaded with DNR by engineering the nano-pore size of pSi.

Oxidized porous silicon particles covalently grafted with daunorubicin as a sustained intraocular drug delivery system.

PURPOSE To test the feasibility of covalent loading of daunorubicin into oxidized porous silicon (OPS) and to evaluate the ocular properties of sustained delivery of daunorubicin in this system. METHODS Porous silicon was heat oxidized and chemically functionalized so that the functional linker on the surface was covalently bonded with daunorubicin. The drug loading rate was determined by thermogravimetric analysis. Release of daunorubicin was confirmed in PBS and excised rabbit vitreous by mass spectrometry. Daunorubicin-loaded OPS particles (3 mg) were intravitreally injected into six rabbits, and ocular properties were evaluated through ophthalmic examinations and histology during a 3-month study. The same OPS was loaded with daunorubicin using physical adsorption and was evaluated similarly as a control for the covalent loading. RESULTS In the case of covalent loading, 67 ± 10 μg daunorubicin was loaded into each milligram of the particles while 27 ± 10 μg/mg particles were loaded by physical adsorption. Rapid release of daunorubicin was observed in both PBS and excised vitreous (~75% and ~18%) from the physical adsorption loading, while less than 1% was released from the covalently loaded particles. Following intravitreal injection, the covalently loaded particles demonstrated a sustained degradation of OPS with drug release for 3 months without evidence of toxicity; physical adsorption loading revealed a complete release within 2 weeks and localized retinal toxicity due to high daunorubicin concentration. CONCLUSIONS OPS with covalently loaded daunorubicin demonstrated sustained intravitreal drug release without ocular toxicity, which may be useful to inhibit unwanted intraocular proliferation.

Porous silicon oxide–PLGA composite microspheres for sustained ocular delivery of daunorubicin

A water-soluble anthracycline antibiotic drug (daunorubicin, DNR) was loaded into oxidized porous silicon (pSiO2) microparticles and then encapsulated with a layer of polymer (poly lactide-co-glycolide, PLGA) to investigate their synergistic effects in control of DNR release. Similarly fabricated PLGA-DNR microspheres without pSiO2, and pSiO2 microparticles without PLGA were used as control particles. The composite microparticles synthesized by a solid-in-oil-in-water emulsion method have mean diameters of 52.33±16.37μm for PLGA-pSiO2_21/40-DNR and the mean diameter of 49.31±8.87μm for PLGA-pSiO2_6/20-DNR. The mean size, 26.00±8μm, of PLGA-DNR was significantly smaller, compared with the other two (P<0.0001). Optical microscopy revealed that PLGA-pSiO2-DNR microspheres contained multiple pSiO2 particles. In vitro release experiments determined that control PLGA-DNR microspheres completely released DNR within 38days and control pSiO2-DNR microparticles (with no PLGA coating) released DNR within 14days, while the PLGA-pSiO2-DNR microspheres released DNR for 74days. Temporal release profiles of DNR from PLGA-pSiO2 composite particles indicated that both PLGA and pSiO2 contribute to the sustained release of the payload. The PLGA-pSiO2 composite displayed a more constant rate of DNR release than the pSiO2 control formulation, and displayed a significantly slower release of DNR than either the PLGA or pSiO2 formulations. We conclude that this system may be useful in managing unwanted ocular proliferation when formulated with antiproliferation compounds such as DNR.

Intravitreal controlled release of dexamethasone from engineered microparticles of porous silicon dioxide.

Dexamethasone is a glucocorticoid that is widely used in the ophthalmic arena. The recent FDA approved dexamethasone implant can provide a three month efficacy but with high rate of drug related cataract and high intraocular pressure (IOP). It seems that higher steroid in aqueous humor and around lens may be associated with these complications based on clinical fact that higher IOP was observed with intravitreal triamcinolone acetonide (TA) than with subtenon TA. We hypothesize that placing a sustained dexamethasone release system near back of the eye through a fine needle can maximize efficacy while mitigate higher rate of IOP rise and cataract. To develop a sustained intravitreal dexamethasone delivery system, porous silicon dioxide (pSiO2) microparticles were fabricated and functionalized with amines as well as carboxyl groups. Dexamethasone was conjugated to pSiO2 through the Steglich Esterification Reaction between hydroxyl of dexamethasone and carboxyl groups on the pSiO2. The drug loading was confirmed by Fourier transform infrared spectroscopy (FTIR) and loading efficiency was quantitated using thermogravimetric analysis (TGA). In vitro release was conducted for three months and dexamethasone was confirmed in the released samples using liquid chromatography-tandem mass spectrometry (LC/MS/MS). A pilot ocular safety and determination of vitreous drug level was performed in rabbit eyes. The drug loading study demonstrated that loading efficiency was from 5.96% to 10.77% depending on the loading reaction time, being higher with longer loading reaction time before reaching saturation around 7 days. In vitro drug release study revealed that dexamethasone release from pSiO2 particles was sustainable for over 90 days and was 80 days longer than free dexamethasone or infiltration-loaded pSiO2 particle formulation in the same setting. Pilot in vivo study demonstrated no sign of ocular adverse reaction in rabbit eyes following a single 3 mg intravitreal injection and free drug level at 2-week was 107.23 ± 10.54 ng/mL that is well above the therapeutic level but only around 20% level of dexamethasone released from OZURDEX(®) (dexamethasone intravitreal implant) in a rabbit eye model. In conclusion, dexamethasone is able to covalently load to the pSiO2 particles and provide sustained drug release for at least 3 months in vitro. Intravitreal injection of these particles were well tolerated in rabbit eyes and free drug level in vitreous at 2-week was well above the therapeutic level.

Surface engineering of porous silicon microparticles for intravitreal sustained delivery of rapamycin.

PURPOSE To understand the relationship between rapamycin loading/release and surface chemistries of porous silicon (pSi) to optimize pSi-based intravitreal delivery system. METHODS Three types of surface chemical modifications were studied: (1) pSi-COOH, containing 10-carbon aliphatic chains with terminal carboxyl groups grafted via hydrosilylation of undecylenic acid; (2) pSi-C12, containing 12-carbon aliphatic chains grafted via hydrosilylation of 1-dodecene; and (3) pSiO2-C8, prepared by mild oxidation of the pSi particles followed by grafting of 8-hydrocarbon chains to the resulting porous silica surface via a silanization. RESULTS The efficiency of rapamycin loading follows the order (micrograms of drug/milligrams of carrier): pSiO2-C8 (105 ± 18) > pSi-COOH (68 ± 8) > pSi-C12 (36 ± 6). Powder X-ray diffraction data showed that loaded rapamycin was amorphous and dynamic drug-release study showed that the availability of the free drug was increased by 6-fold (compared with crystalline rapamycin) by using pSiO2-C8 formulation (P = 0.0039). Of the three formulations in this study, pSiO2-C8-RAP showed optimal performance in terms of simultaneous release of the active drug and carrier degradation, and drug-loading capacity. Released rapamycin was confirmed with the fingerprints of the mass spectrometry and biologically functional as the control of commercial crystalline rapamycin. Single intravitreal injections of 2.9 ± 0.37 mg pSiO2-C8-RAP into rabbit eyes resulted in more than 8 weeks of residence in the vitreous while maintaining clear optical media and normal histology of the retina in comparison to the controls. CONCLUSIONS Porous silicon-based rapamycin delivery system using the pSiO2-C8 formulation demonstrated good ocular compatibility and may provide sustained drug release for retina.

A Novel Approach of Daunorubicin Application on Formation of Proliferative Retinopathy Using a Porous Silicon Controlled Delivery System: Pharmacodynamics.

PURPOSE Proliferative vitreoretinopathy (PVR) is the most common cause of poor visual outcomes in association with retinal detachment surgeries and ocular trauma. Daunorubicin (DNR) has shown the strongest efficacy in proliferation inhibition in vitro. However, clinical studies have shown only mild effect owing to limitations of narrow therapeutic window and short vitreous half-life. METHODS Three milligrams of DNR-loaded particles were intravitreally injected into 18 pigmented rabbits, and vitreous samples were collected up to 84 days for analysis. Thirty-seven rabbits were used for a dose-escalation (1, 3, 6 mg) safety and efficacy study in a rabbit PVR model using a pretreatment design. RESULTS Loading efficiency of DNR was 108.55 ± 12 μg per 1 mg particles. Eighty-four days of follow-up did not reveal any adverse reaction. Pharmacokinetic analysis demonstrated a vitreous half-life of 29 days with a maximum DNR concentration of 178 ng/mL and a minimum concentration of 29 ng/mL at day 84. Daunorubicin-loaded porous silicon (pSi) particles were dosed 8 to 9 weeks before PVR induction, and PVR severity score was dose dependent (Spearman ρ = -0.25, P = 0.0005). Proliferative vitreoretinopathy with tractional retinal detachment was 88% in the control group, 63% in the low-dose group, 14% in the medium-dose group, and 0% in the high-dose group (Cochran-Armitage Trend Test, Z = 8.99, ρ = -0.67, P < 0.0001). CONCLUSIONS Daunorubicin-loaded pSi particles can safely reside in the vitreous for at least 3 months. The pSi-based delivery expanded the therapeutic window of DNR by a factor of 862 and drove down the minimum effective concentration by a factor of 175.

Micelle formulation of hexadecyloxypropyl-cidofovir (HDP-CDV) as an intravitreal long-lasting delivery system

There still is an unmet need for a safe and sustained intravitreal drug delivery system. In this study we are proposing and characterizing a micelle based, clear-media intravitreal drug delivery system using the lipid derivatized nucleoside analog, hexadecyloxypropyl-cidofovir (HDP-CDV, CMX 001). HDP-CDV forms micelles in water and in vitreous supernatant with the critical micelle concentration of 19 μg/mL and 9 μg/mL, respectively at 37 °C. The formed micelles had the average size of 274.7 nm and the Zeta potential of -47.1 mV. Drug release study in the excised rabbit vitreous showed a sustained release profile with a half-life of 2.7 days. The micelle formulation of HDP-CDV demonstrated a good safety profile in two animal species (rabbit and guinea pig) following intravitreal injection. The sustained efficacy was tested in a pretreatment study design and the drug potency was tested in an ongoing herpes simplex virus (HSV-1) retinitis model. The pretreatment studies using single intravitreal injection and later HSV-1 infection revealed at least 9 weeks of vitreous presence and therapeutic level of HDP-CDV, with 71% eyes protection from infection. The treatment study demonstrated that intravitreal administration halted active HSV-1 retinitis in 80% of the infected eyes while cidofovir (CDV) treatment failed to suppress active HSV-1 retinitis. In summary, lipid derivatized nucleoside analogs can be formulated as a micelle intravitreal injection and provides a sustained drug release in vitreous for chronic retinal diseases.

Macular choroidal volume variations in highly myopic eyes with myopic traction maculopathy and choroidal neovascularization.

Purpose: To compare the choroidal volume (CV) between emmetropic and highly myopic eyes, and to assess if the presence of myopic fundus abnormalities, myopic traction maculopathy, or choroidal neovascularization affects the CV. Methods: We retrospectively reviewed imaging studies of 98 eyes of 98 patients who underwent CV measurement on optical coherence tomography. We included 31 emmetropic eyes (Group 1), 36 highly myopic eyes without vitreoretinal or choroidal pathologies (Group 2), 21 highly myopic eyes with traction maculopathy (Group 3), and 10 highly myopic eyes with history of choroidal neovascularization (Group 3). Eyes with chorioretinal atrophy were excluded. Regression analysis was performed to evaluate the correlation between CV and multiple variables. Results: Choroidal volume was lower in Group 2 than in Group 1 (P < 0.001), and in Groups 3 and 4 than in Group 2 (P < 0.001 and P = 0.002, respectively). Age (P = 0.002), axial length (P < 0.001), sex (P = 0.047), staphyloma (P < 0.001), and myopic group (P = 0.05) were independent predictors for the final CV (R2 = 0.645). In highly myopic eyes, CV decreased by 0.32 mm3 for every 10 years and by 0.49 mm3 per millimeter of axial length. Conclusion: Choroidal thinning is present in highly myopic eyes compared with emmetropic eyes, and is related to age, axial length, sex, and staphyloma. However, myopic eyes with coexisting myopic traction maculopathy or history of choroidal neovascularization have more severe thinning, likely leading to insufficient metabolic supplementation for the macula.

Controlled Release of Dexamethasone From an Intravitreal Delivery System Using Porous Silicon Dioxide

Purpose The current study aims to evaluate a porous silicon-based drug delivery system meant for sustained delivery of dexamethasone (Dex) to the vitreous and retina. Methods Dexamethasone was grafted covalently into the pore walls of fully oxidized porous silicon particles (pSiO2-COO-Dex), which then was evaluated for the pharmacological effect of the payload on cultured ARPE19 cells before intravitreal injection. The Dex release profile was investigated in a custom designed dynamic dissolution chamber to mimic the turnover of vitreous fluid in rabbit eyes. Ocular safety, in vivo release, and pharmacodynamics were evaluated in rabbit eyes, and the human VEGF-induced rabbit retinal vascular permeability model. Results Loading efficiency of Dex was 69 ± 9 μg per 1 mg of the pSiO2-COO-Dex particles. Dynamic in vitro release demonstrated a sustained mode when compared to free Dex, with the drug half-life extended by 5 times. The released Dex was unaltered and biologically active. In vivo drug release in rabbit eyes revealed a mode similar to the release seen in vitro, with a vitreous half-life of 11 days. At 2 and 4 weeks after a single intravitreal injection of pSiO2-COO-Dex particles (mean 2.71 ± 0.47 mg), intravitreal 500 ng of VEGF did not induce significant retinal vessel dilation or fluorescein leakage, while these events were observed in the eyes injected with empty pSiO2 particles or with free Dex. The retinal vessel score from fluorescein angiography for the control eyes was double the score for the eyes injected with pSiO2-COO-Dex. No adverse reaction was observed for the eyes injected with drug-loaded pSi particles during the course of the study. Conclusions The porous silicon-based Dex delivery system (pSiO2-COO-Dex) can be administered safely into vitreous without toxicity. Dex release from the porous silicon particles was sustained for 2 months and was effective against VEGF-induced retinal vessel reaction.

Bone Marrow-Derived Cells in Neovascular Age-Related Macular Degeneration: Contribution and Potential Application

Neovascular age-related macular degeneration, characterized by the formation of choroidal neovascularization (CNV), is a predominant cause of serious loss of vision. The pathogenesis of CNV is complex and still imperfectly understood. Prior studies have shown that bone marrow-derived cells (BMC) play a role in CNV. In this review article, we describe the contribution of BMC to CNV development, and discuss the potential use of BMC in the anticipation and treatment of CNV-associated diseases as well as research needs in the future.