Ocular biocompatibility evaluation of POSS nanomaterials for biomedical material applications

Abstract

The tremendous advancement of polyhedral oligomeric silsesquioxanes (POSSs) has been focused on the field of biomaterial applications including tissue engineering, drug delivery, biomedical devices and biosensors. More recently, POSSs have been used in components of ophthalmic biomedical devices, such as contact lenses and intraocular lenses due to their chemical inertness and transparency. A systematic biocompatibility evaluation of POSS nanomaterials is thus essential. Herein, the ocular biocompatibility and cytotoxicity of POSS nanomaterials were investigated both in vitro and in vivo. Three types of commercial POSS nanomaterials with different functional groups were utilized in this research, including aminoethylaminopropylisobutyl-POSS (NH₂-POSS), mercaptopropylisobutyl-POSS (SH-POSS) and octahydroxypropyldimethylsilyl-POSS (HO-POSS). The cellular metabolic activity, membrane integrality, cell apoptosis and oxidative damage were tested on human lens epithelial cells (HLECs) under different concentrations of POSS nanomaterial exposure. The ocular irritation on rabbit eyes was measured as well. The results show that the studied POSS nanomaterials do not exhibit any significant toxicity to cell growth and proliferation in most cases, except for the NH₂-POSS, which decreases the cellular viability at high concentration. All of the POSS nanomaterials slightly induced oxidative stress as a result of an increase in reactive oxygen species (ROS), however they did not generate cell apoptosis. The animal experiment results also show that no acute ocular irritation can be detected after POSS nanomaterial administration. These results indicate the good ocular biocompatibility of the POSS nanomaterials in most cases, which have great potential in ocular biomedical applications.

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Introduction

Since their discovery in 1946 by Scott, polyhedral oligomeric silsequioxanes (POSSs) have gained increasing attention due to their thermal, mechanical and electrical properties.^1,2 POSSs are a class of cage-shaped inorganic–organic hybrid molecules which has grown dramatically in recent years. Their chemical structure is a formula unit between SiO₂ (silica) and R₂SiO (silicone). They follow the typical formula (RSiO_3/2)_n, where n can be an even integer 6, 8, 10 (denoted as T6, T8, T10). Among them, the general POSS molecules (RSiO_3/2)₈ have been the most prevalent system investigated and are broadly deemed as the smallest silica at 1–3 nm. POSSs are really hybrid inorganic–organic chemical materials that consist of an inorganic silica core (0.53 nm) and organic substituent groups, i.e., R-groups which possibly are organic functional groups such as an alkyl, acrylate, sulfonate, amine, hydrogen, hydroxyl, methyl, and so on. The presence of different R-groups subsequently results in complicated and uncontrollable functions.^3–5

A mass of research has shown that POSS molecules incorporated as building blocks into polymers enhanced glass-transition temperatures, thermal stability, antimicrobial properties, mechanical stiffness and electrical properties.^4,6,7 Incorporating POSSs can improve oxidation resistance, and reduce flammability, inflammatory reactions and oxygen permeability, which are the main advantages for their biological applications.⁸ Thus, it is not surprising that POSS polymers have been widely applied in various fields, especially biomedical applications including tissue engineering, drug delivery, biomedical devices and biosensors.^5,9 POSS nanomaterials integrated into poly(carbonate-urea) urethane (PCU) have been successfully used in the first synthetic trachea in the world, which is the first known “in-man” implant utilising POSS.¹⁰ Vascular grafts produced from POSS-PCU implanted into senescent sheep were assessed to have a 64% patency rate.¹¹ A novel lacrimal drainage conduit constructed with POSS-PCU offered good compatibility to resolve a blockage of the tube in lacrimal surgery.¹² What’s more, POSSs are considered as attractive candidates for ophthalmic applications such as in contact lenses and intraocular lenses (IOLs), in terms of their chemical inertness, transparency and biological properties.^3,12 Previous research has found that POSS materials are able to form higher transparency materials which means that they have good light transmission.^13,14 The light transmission of POSSs is suitable for optical materials especially in contact lenses and IOLs.¹⁵ In our previous study, POSS containing composite materials exhibited better transparency and good thermodynamic stability suggesting their potential for use as IOL materials.^14,16

The human eye is a sophisticated and sensitive organ. With the increasing potential of POSS nanomaterials in ocular biomaterials,^14–16 the ocular biocompatibility of POSS nanomaterials should be systematically evaluated. Three kinds of POSS nanomaterials with different functional groups, including amino groups, thiol groups and hydroxyl groups, were taken to investigate the ocular biocompatibility in this research. Human lens epithelial cells (HLECs) play important roles in ocular physiological function. HLECs are located in the anterior surface of the crystalline lens, and are critical for lens formation, physiology and transparency, as well as the key reason for cataractogenesis.^17,18 The dysfunction of HLECs may result in the posterior capsular opacification after IOL implantation. So HLECs were used for the cytotoxicity measurement of POSS nanomaterials. The toxicity, cellular membrane integrity, apoptosis and oxidative stress of HLECs, as well as the ocular irritation on rabbit eyes under POSS nanomaterial exposure were investigated for a systematic evaluation of the ocular biocompatibility.

Experimental section

Materials

Octahydroxypropyldimethylsilyl-polyhedral oligomeric silsesquioxane (HO-POSS, M_w = 1482.60 g mol⁻¹), aminoethylaminopropylisobutyl-polyhedral oligomeric silsesquioxane (NH₂-POSS, M_w = 917.65 g mol⁻¹), mercaptopropylisobutyl-polyhedral oligomeric silsesquioxane (SH-POSS, M_w = 891.63 g mol⁻¹), and 2,7-dichlorodihydro-fluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich (United States). Dulbecco’s modified Eagle’s medium (DMEM): F12 (1 : 1), fetal bovine serum (FBS), Hank’s balanced salt solution (HBSS), 0.05% trypsin-EDTA and penicillin-streptomycin were bought from Life technology (United States). Cell counting kit-8 (CCK-8), lactate dehydrogenase release assay kit (LDH assay), and Hoechst staining kit were provided by the Beyotime Institute of Biotechnology (China). Human lens epithelial cell lines (HLE B3, CRL-11421™) originated from the American Type Culture Collection (ATCC). 200 mg mL⁻¹ of POSS nanomaterial stock solution was dissolved in tetrahydrofuran, and diluted by cell culture medium as used.

Cell culture

HLECs were cultivated in DMEM: F12 (1 : 1) media which was supplemented with 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin solution. The cells were maintained in an incubator with humidified air with 5% CO₂ at 37 °C, replacing the media every two days. When the cells were grown to 80% confluence, 0.05% trypsin-EDTA was utilized for cell passage.

Cell membrane integrality assay

Cell membrane integrality was measured using a LDH leakage measurement, according to the kit protocol. Briefly, after exponentially growing cells were collected, 5 × 10³ cells were seeded into 96-well plates and incubated at 37 °C overnight. The next day, dose- and time-dependent surveys of the LDH assay were carried out by treating with 5, 10, 50, 100, 150, 200 mg L⁻¹ of NH₂-POSS, SH-POSS, HO-POSS for 24 h, 48 h, 72 h. One hour before the appointed time, 10% LDH cell lysis solutions were added to the cells without POSSs, which were regarded as the total releasing samples. The untreated cells (without lysis solution and POSS treatment) were regarded as spontaneous releasing samples. The plates were then centrifuged at 300g for 5 minutes. Aliquots of cell supernatants without cells were transferred to new 96-wells plates, mixed with LDH working solution at room temperature in the dark and the optical density (OD) was read using a microplate reader at 490 nm. The rates of LDH release (%) were calculated as follows: LDH release (%) = (OD_test − OD_spontaneity)/(OD_total − OD_spontaneity) × 100%.

Cell viability assay

Cell viability on the basis of mitochondrial function was measured by a CCK-8 assay. HLECs were plated into 96-well plates at 5 × 10³ cells per well and cultured overnight. Afterwards, the primary media was discarded and fresh media was added which contained different concentrations of POSS (NH₂-POSS, SH-POSS, HO-POSS, as shown in Fig. 1). The concentrations of POSS nanomaterials were 5, 10, 50, 100, 150, 200 mg L⁻¹, respectively. Cells without treatment were considered as the control. At the end of the incubation time (24 h, 48 h, 72 h), the cells were washed twice with PBS and incubated with CCK-8 for 2 h at 37 °C. The OD was monitored using a microplate reader at 450 nm. The cell viability (%) was calculated using the following equation: cell viability (%) = OD_test/OD_control × 100%.

Fig. 1 The schematic structure of POSS (A), NH₂-POSS (B), SH-POSS (C) and HO-POSS (D).

ROS secretion

The intracellular ROS secretion was detected by applying fluorescence probes DCFH-DA as previously reported.¹⁹ HLECs seeded with a density of 5 × 10⁴ cells per mL were plated on 6-well plates overnight. Then, the cells were incubated with 200 mg L⁻¹ of different POSS nanomaterials (NH₂-POSS, SH-POSS and HO-POSS, respectively) for 24 h, followed by DCFH-DA staining at 37 °C for 30 minutes. The fluorescent images were taken using a fluorescence microscope.

Cell apoptosis

Apoptosis occurred by fragmentation or condensation of nuclei, and as a result the normal cells showed up as blue, while the apoptosis cells showed a uniform dispersion of fluorescent particles or were brighter, even whitish, using Hoechst staining. A Hoechst 33258 staining kit was used to determine HLEC apoptosis under POSS exposure. Briefly, 5 × 10⁴ cells of the HLEC line were grown in 24-well plates for 24 h. At the final concentration of 200 mg L⁻¹ of POSS, the media solutions were replaced and induced for 24 h. Cells were stained using the Hoechst staining kit and visualized using a fluorescence microscope.

Acute ocular irritation

The approval of the Laboratory Animal Ethics Committee of Wenzhou Medical University for animal study was obtained. Japanese white rabbits with weights from 2.0–3.0 kg were obtained from the Animal Administration Center of Wenzhou Medical University and raised at 22 ± 3 °C, in 40 to 70% humidity environments. The acute ocular irritation of POSS nanomaterials was carried out in accordance with the GBZ/T240.5-2011 and Organization for Economic Cooperation and Development (OECD) Test Guidelines 405. After a careful health check, the rabbits’ eyes were instilled with 100 μL of POSS nanomaterial drops (200 μg mL⁻¹) on the right eye. The left eyes were dropped with normal saline as a control. The rabbits were weighed, and ophthalmological observations were performed on the cornea, iris or conjunctiva at 0 h, 1 h, 6 h, 24 h, 48 h, and 72 h using fluorescein staining and slit lamp microscopes.

Statistical analysis

All data are presented as the average ± standard deviations (SD) (n ≥ 3). Statistical significance (p < 0.05) was analyzed with Statistical Product and Service Solutions (SPSS) Software using one-way or two-way ANOVA analysis.

Results and discussion

The advances in nanomedicine have proposed novel therapeutics and diagnostics, which can potentially revolutionize current medical practices. POSSs with distinctive nano-cage structures consisting of an inner inorganic framework of silicon and oxygen atoms, and an outer shell of organic functional groups, are one of the most promising nanomaterials for medical applications.^20–23 Enhanced physicochemical properties and biocompatibility have resulted in the development of a wide range of nanocomposite POSS copolymers for biomedical applications, such as the development of biomedical devices, tissue engineering scaffolds, drug delivery systems, dental applications, and biological sensors in the past few years.¹² More recently, POSS nanomaterials were found to be prospective materials for ophthalmic applications such as contact lenses and IOLs due to their chemical inertness, transparency and oxygen permeability.^14–16 So it is necessary to investigate the ocular biocompatibility of POSS nanomaterials deeply. Although in vitro cytotoxicity assays including viability, cell membrane integrity, or apoptosis provide information on the biocompatibility of POSS nanomaterials, they can’t reflect the in vivo response of the nanomaterials, as each test only presents one endpoint of cell response. A combination of several tests may provide more detailed information of the biocompatibility of the nanomaterials.

Herein, the ocular biocompatibility of POSS nanomaterials with different functional groups, including amino, hydroxyl and thiol groups (as shown in Fig. 1), was systematically assessed by testing the damage to the ocular cell membrane and the mitochondrial dehydrogenase viability or ROS secretion in cytoplasm and apoptosis via the nucleus, as well as the animal ocular surface irritation.

The HLEC cell membrane integrity under POSS nanomaterial exposure was investigated using a LDH assay. When negative effects happened, the cells would result in membrane rupture, and for that reason cytoplasmic enzymes leaked out which included stable LDH.²⁴ The released LDH activity was determined by a coupled enzymatic reaction via the LDH kit. Fig. 2 shows that the LDH release rate against NH₂-POSS, SH-POSS and HO-POSS at different concentrations and culture times. No time dependence on cell membrane integrity was observed. The LDH release rate is in the range of ±5% even after incubation in 200 mg mL⁻¹ of POSSs for 72 h, suggesting low cell membrane damage of the POSSs.

Fig. 2 LDH release rate of NH₂-POSS, SH-POSS and HO-POSS (5–200 mg mL⁻¹) on HLECs at 24 h, 48 h and 72 h as measured by the LDH assay.

The HELC viability in POSS-incorporated culture medium was measured by a CCK-8 assay. The same as the MTT assay,^25,26 the CCK-8 assay is based on the reduction of a water-soluble tetrazolium salt to formazan by mitochondrial dehydrogenase. As shown in Fig. 3, the cell viabilities of SH-POSS and HO-POSS were higher than 90% for three days in the tested concentrations, indicating the high cell viability of HLECs under SH-POSS and HO-POSS exposure. However, NH₂-POSS had different effects on the cells. The cell viability of HLECs under NH₂-POSS exposure was above 100% when the nanomaterial concentration was under 10 mg L⁻¹ in the observation periods. Whereas the cell viability significantly decreased to 47.6%, 44.0%, 35.8% and 24.0% in concentrations of 50 mg L⁻¹, 100 mg L⁻¹, 150 mg L⁻¹ and 200 mg L⁻¹ at 24 h. The prolonged culture time induced a lower cell viability. The viability decreased to 12% in a concentration of 200 mg L⁻¹ when cultured for 72 h. These results demonstrated that SH-POSS, HO-POSS or low doses of NH₂-POSS (≤10 mg L⁻¹) present low cytotoxicity to HELCs, whereas high doses of NH₂-POSS (≥50 mg L⁻¹) have noticeable cytotoxicity to HLECs.

Fig. 3 Cell viability of NH₂-POSS, SH-POSS and HO-POSS on HLECs.

ROS are chemically reactive molecules containing oxygen, including oxygen ions, superoxide anions, peroxides and hydrogen peroxide. ROS are formed as a natural byproduct of the normal metabolism of oxygen and have important roles in cell signaling and homeostasis.²⁷ However, during times of environmental stress, ROS levels can increase dramatically, which may result in significant damage to cell structures.²⁷ This is known as oxidative stress. Herein, the POSS nanomaterial induced oxidative stress on HLECs is elucidated via a DCFH-DA staining assay.²⁸ The method operates on the basis of the oxidization of cell permeable DCFH-DA without any fluorescence to the highly fluorescent DCF upon reaction with cellular ROS.²⁹ The cells were incubated with POSS nanomaterials with different functional groups for 24 h, followed by DCFH-DA staining and fluorescence microscope observation. As shown in Fig. 4A, low levels of ROS secretion are presented in native cells, whereas the POSS nanomaterial exposure induced the ROS up-regulation in the cytoplasm (Fig. 4B–D). It can be observed that the cells exposed with HO-POSS have less ROS secretion, compared with those exposed with NH₂-POSS or SH-POSS.

Fig. 4 Fluorescence micrographs of DCFH-DA stained HLECs (A), and cells incubated with 200 mg mL⁻¹ NH₂-POSS (B), HO-POSS (C) or SH-POSS (D), for indicating the intracellular ROS level.

To further investigate the cytotoxicity of the POSS nanomaterials, the cell nucleus damage was investigated. The nucleus damage of the cells may cause cell apoptosis, which can be defined by the nuclear morphology using Hoechst 33258 staining. As shown in Fig. 5, the regular cells have regular contours and homogenous nuclei showing normal blue staining, but the apoptotic cells exhibit nuclear pyknosis, condensed chromatin and fragmented nuclei, which show up as enhanced bright blue signals (arrows in Fig. 5). The control cells without POSS exposure exhibit rare hyper-condensation and shrinkage of nuclei, with an apoptosis rate of 2.3%, suggesting the basic level of apoptosis. When treated with 200 mg L⁻¹ of POSS nanomaterials for 24 h, a slight increase in apoptotic cells is found. The apoptosis rates are in the range of 3.1% to 5.9%, which are at a low cell apoptosis level. This result indicates that although the POSS nanomaterial incubation may slightly increase nucleus damage, the cell apoptosis rates of different functional groups are in the low level (<5%).

Fig. 5 Fluorescence micrograph of Hoechst 33258 staining of HLECs (A) and cells incubated with 200 mg L⁻¹ NH₂-POSS (B), HO-POSS (C) and SH-POSS (D) for 24 h (white arrows indicate cell apoptosis) (200×).

The results of the cytotoxicity assessments are summarized in Table 1. When nanomaterials are exposed to cells and cause damage, the first step is the damage to the cell membrane. The nanomaterials may enter cells via endocytosis, causing some damage to the cell membrane integrity. The LDH releasing assay is a sensitive target for membrane integrity. The damage of the membrane may cause the release of LDH into the cell culture medium. As shown in Fig. 2, very slight damage to the cell membrane is detected when POSS nanomaterials are incubated with HLECs. No matter if the POSS functional groups are amino groups, thiol groups, or hydroxyl groups, the LDH release rate is lower than 5%, which indicates the non-cytotoxicity to the cell membrane. However, after endocytosis, POSSs may cause some damage to cellular pathways in cytoplasm. The induced ROS secretion is up-regulated after POSS incubation, which may further induce cell dysfunction. The generation of ROS has been reported as one of the key molecular mechanisms of cellular responses and a main factor in determining toxicity.³⁰ Oxidative stress induced by in vitro exposure to nanoparticles can lead to the production of ROS.³¹ However, if the oxidative stress is beyond the level of protective mechanisms, cell death will be programmed via apoptosis, autophagy or necrosis.³² As expected, the ROS level of HLECs with the three POSSs is increased evidently. The secretion of ROS when cells are exposed with nanomaterials may be due to cell self-protection.³³ As a result, although all of the three kinds of POSS nanomaterials induce the ROS up-regulation, not all of them cause strong cytotoxicity. Only the amino group-ended POSS shows an evident decrease to cell viability. The cytotoxicity of the NH₂-POSS on HLECs shows a time dependent or concentration dependent manner. The low concentration does not show evident toxicity to HLECs, whereas high concentrations of NH₂-POSS render strong cytotoxicity. The cytotoxicity increases with the incubation time increasing. The activity of mitochondrial dehydrogenase decreases to 12% when incubated with 200 mg L⁻¹ NH₂-POSS and cultured for 72 h. However, the viability of the HLECs is as high as 90% to 110% of the control when exposed with HO-POSS or SH-POSS. Surface groups can have different functions if they are hydrophilic or hydrophobic, cationic or anionic.³⁴ Stronger cytotoxicity of amino-terminated materials is also presented in other particles.³⁵ In aqueous solution, amino groups show a higher degree of cationic charge than thiol groups, while hydroxyl groups are neutral. Positive charge constructs show stronger cytotoxicity than negative ones.³⁶ On one hand, positive groups are electrostatically attracted to the negatively charged plasma membrane, and can thereby interfere with cellular natural activity.³⁷ On the other hand, positive ones are often easily taken up but higher uptakes are related to greater toxic effects.³⁸ Although NH₂-POSS is found to have strong cytotoxicity on cell viability, it does not show greater damage to the cell nucleus, compared with HO-POSS and SH-POSS at the same concentration. According to the Hoechst staining, it is observed that significant apoptosis was found neither in the POSS groups nor in the control group. Only a slight increase of nuclear pyknosis is observed in all of three kinds of POSS. The induced cell apoptosis rate is below 6% in all the cases.

Table 1 Summary of the damage of NH₂-POSS, SH-POSS and HO-POSS to HELCs^a

Group	Cell membrane integrity damage	Mitochondrial dehydrogenase viability	Oxidative stress	Nucleus damage
a (+) indicates POSSs with a significant response, while (−) indicates POSSs without a significant harmful response.
NH2-POSS	−	++	+	−
SH-POSS	−	−	+	−
HO-POSS	−	−	+	−

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In the present study, a notable up-regulation of ROS is found with POSS nanomaterial exposure whereas only NH₂-POSS at high concentration is found to have strong cytotoxicity in cell viability, let alone that almost no damage to the cell membrane and the nucleus is detected in all of the investigated POSS nanomaterials. Considering that the toxic pathway of the nanoparticles may attribute to the cell autophagy or necrosis rather than apoptosis,^39,40 the cytotoxicity of POSSs to HLECs may cause cell death through autophagy or necrosis instead of apoptosis.²⁶ The cytotoxicity mechanism of POSS is that ROS is rising under oxidative stress as an initiating response which may not result in apoptosis but rather protect cells from damage and further studies are required to verify the mechanism.²⁷

For ocular biomedical application purposes, the acute ocular irritation of POSS nanomaterials on rabbits is further carried out. Typically, the rabbits’ eyes were instilled with 100 μL of POSS drops (200 μg mL⁻¹) on the right eye. The left eyes were dropped with normal saline as a control. The rabbits were weighed, and ophthalmological observations were performed on the cornea, iris or conjunctiva at 0 h, 1 h, 6 h, 24 h, 48 h, and 72 h using fluorescein staining and a slit lamp microscope. Fig. 6 shows the weight changes after the POSS nanomaterial administration. It is obvious that the weights of the animals do not decrease in the experimental periods. The slit lamp observation results (Fig. 7) show that neither acute inflammation nor cornea epithelial lesions are detected after POSS nanomaterial exposure for three days. No significant irritation on the cornea, iris or conjunctiva is found in all of the experimental animal eyes when administered with all three kinds of POSS nanomaterials. These results show that the POSS nanomaterials have excellent in vivo biocompatibility with ocular tissues.

Fig. 6 The weight changes of rabbits after ocular irritation.

Fig. 7 Slit lamp images of the rabbit eyes with POSS or normal saline drop ocular surface administration and fluorescein stained at 0, 1, 24, 48 and 72 hours.

Conclusions

The ocular biocompatibility of POSSs was systematically assessed by testing the POSS nanomaterial damage to the ocular cell membrane, mitochondrial dehydrogenase viability or ROS secretion in cytoplasm and apoptosis via the nucleus, as well as the animal ocular surface irritation. The results show that the POSS nanomaterials do not cause damage to the HLEC membrane and nucleus, whereas they will notably up-regulate the secretion of ROS. The up-regulation of ROS does not directly relate with the cell viability or apoptosis. Only high concentrations of NH₂-POSS present cytotoxicity to cell viability, whereas HO-POSS and SH-POSS render good cell viability even at high concentrations and with incubation for a long time. The in vivo results show that neither acute inflammation nor cornea epithelial lesions are detected after POSS nanomaterial exposure. No significant irritation of the cornea, iris or conjunctiva was found in all of the experimental animal eyes. These results show the good ocular biocompatibility of POSS nanomaterials except for NH₂-POSS in high concentration, indicating that POSS nanomaterials may be a good alternative ocular biomedical material in most cases.

Acknowledgements

Financial support from the National Natural Science Foundation of China (51203120, 81271703, 51403158), Science & Technology Program of Wenzhou (Y20140177) and Medical & Health Technology Program of Zhejiang Province (2013KYA133, 2014KYA149) are greatly acknowledged.

Notes and references

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Footnote

† These authors contributed equally.

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