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.

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.

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.

Many chorioretinal diseases are chronic and need sustained drug delivery systems to keep therapeutic drug level at the disease site. Many intravitreal drug delivery systems under developing do not have mechanism incorporated for a non-invasive monitoring of drug release. The current study prepared rugate porous silicon (pSi) particles by electrochemical etching with the current frequency (K value) of 2.17 and 2.45. Two model drugs (rapamycin and dexamethasone) and two drug-loading strategies were tested for the feasibility to monitor drug release from the pSi particles through a color fundus camera. The pSi particles (k=2.45) with infiltration loading of rapamycin demonstrated progressively more violet color reflection which was negatively associated with the rapamycin released into the vitreous (r=-0.4, p<0.001, pairwise). In contrast, pSi with K value of 2.17 demonstrated progressive color change toward green and a weak association between rapamycin released into vitreous and green color abundance was identified (r=-0.23, p=0.002, pairwise). Dexamethasone was covalently loaded on to the fully oxidized pSi particles that appeared in vitreous as yellow color and fading over time. The yellow color decrease over time was strongly associated with the dexamethasone detected from the vitreous samples (r=0.7, p<0.0001, pairwise). These results suggest that engineered porous silicon particles may be used as a self-reporting drug delivery system for a non-invasive real time remote monitoring.

Statement of significance

The current study, for the first time, demonstrated proof of concept that engineered porous silicon photonic crystal may deliver therapeutics in a controlled fashion while at the same time might offer a noninvasive remote monitoring of its payload release in a living eye. Porous silicon photonic crystal changes color which is in association with its payload release into vitreous. With further optimization, the color change may be harnessed to inform eye care professionals of real time drug concentration in the eye and allow them to make informed decision to re-dose the patients.

The 22q11.2 deletion syndrome (22q11.2DS) results from non-allelic homologous recombination between low-copy repeats termed LCR22. About 60%-70% of individuals with the typical 3 megabase (Mb) deletion from LCR22A-D have congenital heart disease, mostly of the conotruncal type (CTD), whereas others have normal cardiac anatomy. In this study, we tested whether variants in the hemizygous LCR22A-D region are associated with risk for CTDs on the basis of the sequence of the 22q11.2 region from 1,053 22q11.2DS individuals. We found a significant association (FDR p < 0.05) of the CTD subset with 62 common variants in a single linkage disequilibrium (LD) block in a 350 kb interval harboring CRKL. A total of 45 of the 62 variants were associated with increased risk for CTDs (odds ratio [OR) ranges: 1.64-4.75). Associations of four variants were replicated in a meta-analysis of three genome-wide association studies of CTDs in affected individuals without 22q11.2DS. One of the replicated variants, rs178252, is located in an open chromatin region and resides in the double-elite enhancer, GH22J020947, that is predicted to regulate CRKL (CRK-like proto-oncogene, cytoplasmic adaptor) expression. Approximately 23% of patients with nested LCR22C-D deletions have CTDs, and inactivation of Crkl in mice causes CTDs, thus implicating this gene as a modifier. Rs178252 and rs6004160 are expression quantitative trait loci (eQTLs) of CRKL. Furthermore, set-based tests identified an enhancer that is predicted to target CRKL and is significantly associated with CTD risk (GH22J020946, sequence kernal association test (SKAT) p = 7.21 × 10^-5) in the 22q11.2DS cohort. These findings suggest that variance in CTD penetrance in the 22q11.2DS population can be explained in part by variants affecting CRKL expression.