PLAâPEG Micelles Loaded with a Classic Vasodilator for Oxidative

Letter pubs.acs.org/journal/abseba

Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

PLA−PEG Micelles Loaded with a Classic Vasodilator for Oxidative Cataract Prevention Lu Xu, Wen-Xiu Qiu, Wen-Long Liu, Chi Zhang, Mei-Zhen Zou, Yun-Xia Sun,* and Xian-Zheng Zhang* Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072, People’s Republic of China

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S Supporting Information *

ABSTRACT: The only treatment for cataract in clinic is the clouded lens removal combined with artificial lens implantation. In this study, nifedipine (NFP), a classic vasodilator, was loaded in a U.S. FDA-approved polymer PLA−PEG to form NFPloaded PLA−PEG micelles as a novel eye drop to prevent oxidative cataract formation and progression at the early stage. The NFP-loaded PLA−PEG micelles not only showed satisfactory biocompatibility and bioavailability, but also efficiently improved the anticataract ability through the inhibition of extracellular calcium ions influx. This study may provide a new insight into the development of cataract treatment. KEYWORDS: micelles, calcium channel blocker, oxidative stress, cataract treatment, eye drop

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Although researchers have designed various kinds of watersoluble molecules or extracted the effective components from traditional Chinese herbs including epigallocatechin gallate, Nacetylcarnosine, honokiol, and tetramethylpyrazine, and used these drugs as eye drops to treat cataract, it would take a long time for these drugs to be applied in clinic due to the lack of clinical trial reports.11−14 Nifedipine (NFP) is a classic, potent, long-acting vasodilator that has been widely used to relieve angina pectoris symptom in clinical treatment.15 Moreover, as the calcium channel blocker, NFP may have the potential to play an important role in the prevention of oxidative cataract by limiting the extracellular calcium influx.16 Clinically, topical ocular eye drops are the most commonly used in ocular drug treatment.17 After administration, the drug in eye drops would permeate the cornea and mix with aqueous humors that surround the lens.18 The clearance rate of drug from aqueous humor is depending on the hydrophilicity of drug, and the clearance rate of lipophilic drugs is faster than that of hydrophilic drugs.19 In this study, NFP was used as an ideal anticataract drug. To improve the solubility and retention time of NFP in aqueous humor, PLA−PEG, a FDA-approved amphiphilic diblock copolymer with good biocompatibility and biodegradability was utilized to load NFP and to form NFP-loaded PLA−PEG nanosized micelles.23 As illustrated in Scheme 1, the NFP-

isual impairment is one of the major health problems across the world. It is estimated that approximately 246 million people are suffering from low vision. Among them, 39 million people are blind. Globally, more than 51% of blindness is related to cataracts.1 Currently, the only available treatment for cataract is the surgical remedy using phacoemulsification cataract extraction combined with artificial lens implantation.2 However, because of the lack of advanced medical equipment and professional ophthalmologists, the cure rate in some developing countries was not as high as that in the developed countries.3 Thus, alternative treatments with low costs and simple administration methods are urgently needed for cataract prevention and treatment. Generally, the causes of cataract include aging, heredity, physical damage, alimentary deficiency, immune abnormalities, and metabolic disorders.4 Among various cataract categories, oxidative cataract is the most common one, which often associates with the increased intraocular oxidative stress.5,6 Normally, the intraocular oxidative stress is resulted from drug side effect, alcohol consumption, and the UV exposure from inevitable sun light.7 The increased oxidative environment around lens epithelial cells would induce the flow of extracellular calcium ions into the cytoplasm through calcium ion channels, which disturbs mitochondria metabolism, activates apoptosis pathway, and finally accelerates cell death.8,9 Meanwhile, phosphorylation and dephosphorylation of numerous proteins in the lens epithelial cells would also be influenced, resulting in the structure alteration, aggregation, and sedimentation of protein and cataract formation.10 © XXXX American Chemical Society

Received: September 11, 2018 Accepted: January 9, 2019 Published: January 9, 2019 A

DOI: 10.1021/acsbiomaterials.8b01089 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Letter

ACS Biomaterials Science & Engineering

Scheme 1. Schematic Illustration of HLECs’ Response to the Oxidative Stress with and without the Protection of NFP-Loaded PLA−PEG Micelles

loaded PLA−PEG micelles would prevent the extracellular calcium influx by blocking the related channels on cell membranes, thus, the mitochondrial apoptosis pathway would not be activated. The denaturation, aggregation, or precipitation of intracellular functional protein could be prevented, and cells would be protected from shrinkage or even death under the oxidative stress. As a result, the cataract formation and progression could be effectively suppressed. Moreover, the NFP-loaded PLA−PEG micelles are convenient for transportation, conservation and administration. This work would not only supply a new application field for NFP, but also provide a new approach in the treatment of cataract. PLA−PEG is an amphiphilic diblock copolymer that could assemble to form micelles to load insoluble NFP in aqueous media through hydrophobic forces. The hydrodynamic size of PLA−PEG micelles measured by dynamic light scatting was around 99.8 nm (Figure 1A) with a narrow size distribution (PDI = 0.176), and the size increased to about 224.3 nm (Figure 1B) when the hydrophobic NFP was loaded, with no obvious change in PDI value (PDI = 0.132). The TEM image showed NFP-loaded PLA−PEG micelles with a spherical shape were well dispersed (Figure 1C). The diameter of NFP-loaded PLA−PEG micelles measured by TEM was about 42 nm, which was smaller than the hydrodynamic diameter evaluated by DLS due to the sample shrinkage under vacuum environment.21,22 The loading capacity of PLA−PEG micelles was 0.96%. The CMC value of PLA−PEG was 10.75 mg/L (Figure 1D), which is much lower than the working concentration (735.7 mg/L) of it, indicating the stability of NFP-loaded PLA−PEG micelles. The in vitro drug release study (Figure 1E) showed the stable release of NFP-loaded in micelles during the first 6 h, and more than 50% of the drugs were released in this stage. Moreover, with the prolonging of incubation time, about 80% of drugs were sustained released in the following hours. This drug releasing behavior would guarantee the effective drug concentration and enough treating times around the nidus. H2O2 was used as an oxidative agent to verify the ability of NFP-loaded PLA−PEG micelles to protect the HLECs from oxidative stress. The HLECs cell viability was evaluated by MTT assay (Figure 1F). With increasing of the H2O2 concentration from 11 μM to 200 μM, the viability of cells incubated with pure H2O2 sharply decreased from 100% to

Figure 1. Characterization of NFP-loaded PLA−PEG micelles. (A) Hydrodynamic particle size of PLA−PEG micelles. (B) Hydrodynamic particle size of NFP-loaded PLA−PEG micelles. (C) TEM image of NFP-loaded PLA−PEG micelles after staining with 1% uranyl acetate, the scale bar is 200 nm. (D) Critical micelle formation concentration (CMC) of NFP-loaded PLA−PEG micelles. (E) The drug release behavior of NFP-loaded PLA−PEG micelles in vitro, the inner figure represents the release behavior in the first 6 h. (F) HLECs viability under various H2O2 concentration with different treatments, the concentration of NFP and NFP-loaded PLA−PEG micelles are all 20 μM. (* = P < 0.05, ** = P < 0.01, *** = P < 0.001).

21%. However, when cells were incubated with NFP-loaded PLA−PEG micelles in the presence of H2O2 (NFP-loaded PLA−PEG micelles + H2O2), ∼90% of cells could maintain their viability even after 24 h. Meanwhile, we found that the viability of cells incubated with NFP in the presence of H2O2 (NFP + H2O2) was not as high as that incubated with NFPloaded PLA−PEG micelles + H2O2. The increased viability of B


Letter


Figure 2. (A) Fluorescence of calcium ion probe in HLECs with various treatments: (a) untreated, (b) 20 μM NFP-loaded PLA−PEG micelles +200 μM H2O2, (c) 20 μM NFP + 200 μM H2O2, (d) 200 μM H2O2, the scale bar is 100 μm. (B) The mean fluorescence intensity of calcium ion probe of HLECs with different treatments. (* = P < 0.05, ** = P < 0.01, *** = P < 0.001). (C) Western blot assay of caspase-3 and cytochrome c expressed by HLECs with various treatments.

Figure 3. (A) The intracellular ROS level in HLECs cells after different treatments: (a) untreated, (b) 20 μM NFP-loaded PLA−PEG micelles +200 μM H2O2, (c) 20 μM NFP + 200 μM H2O2, (d) 200 μM H2O2. (B) Live−dead cell staining of HLECs cells after different treatments: (a) untreated, (b) 20 μM NFP-loaded PLA−PEG micelles +200 μM H2O2, (c) 20 μM NFP + 200 μM H2O2, (d) 200 μM H2O2. The scale bar is 100 μm.

cells incubated in NFP-loaded PLA−PEG micelles + H2O2 may be due to the improved hydrosolubility, stability, and bioavailability of NFP.20 Moreover, the concentration of PLA− PEG and NFP used in this study were 735.7 mg/L and 20 μM, respectively, which would not cause obvious cytotoxity to HLECs (Figure S1A, B).

The intracellular calcium ion concentration reflects the ability of NFP to prevent extracellular calcium ions influx under oxidative stress. After being incubated with NFP or NFP-loaded PLA−PEG micelles for 24 h, cells were stained with intracellular calcium ion probe, and the green fluorescence of probe Fluo-4/AM in each group was observed C


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Figure 4. (A) JC-1 staining of HLECs with different treatments: (a) untreated, (b) 20 μM NFP-loaded PLA−PEG micelles +200 μM H2O2, (c) 20 μM NFP + 200 μM H2O2, (d) 200 μM H2O2. Red fluorescence indicates the maintenance of mitochondrial membrane potential and green fluorescence stands for the loss of mitochondrial membrane potential. The scale bar is 100 μm. (B) The anticataract experiment in extracted rat lens: (a) untreated, (b) 20 μM NFP-loaded PLA−PEG micelles +200 μM H2O2, (c) 20 μM NFP + 200 μM H2O2, (d) 200 μM H2O2. (C) H&E staining of lenses in different treated groups: (a) untreated, (b) 20 μM NFP-loaded PLA−PEG micelles +200 μM H2O2, (c) 20 μM NFP + 200 μM H2O2, (d) 200 μM H2O2. The scale bar is 100 μm.

under fluorescence microscopy. As shown in Figure 2A, negligible fluorescence could be detected in the normal cells (Figure 2A.a). In contrast, strong fluorescence was noted from cells incubated with 200 μM of H2O2 (Figure 2A.d), indicating that numerous extracellular calcium ions flowed into cytoplasm as a response to the high oxidative stress. Besides, in NFP + H2O2 treated cells, the fluorescence intensity decreased accordingly (Figure 2A.c), but the calcium influx could not be prevented completely due to the poor bioavailability of hydrophobic structure of NFP. However, in NFP-loaded

PLA−PEG micelles + H2O2 treated cells, no obvious fluorescence could be observed (Figure 2A.b). The results demonstrated that NFP as a calcium channel blocker could effectively restrained the calcium inflow when entrapped by the PLA−PEG micelles. Moreover, the fluorescence intensity was further quantitatively determined through flow cytometry. As shown in Figure 2B, the mean fluorescence intensity quantified in H2O2 group was about 5.54 × 105, while the value reduced to 4.65 × 105 with addition of free NFP. However, in NFP-loaded PLA− D


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under an inverted fluorescence microscopy. In normal cells (Figure 4A.a), JC-1 could accumulate to form red fluorescence J-aggregates in a mitochondrion with high △Ψm. On the contrary, the probe could not accumulate in mitochondria but exist as a green fluorescence J-monomer in cytoplasm when △Ψm depolarized. As shown in Figure 4A.d, numerous green fluorescence and negligible red fluorescence could be observed, indicating that almost all the mitochondrial membrane potentials were depolarized in 200 μM of H2O2 treated cells. However, strong red fluorescence was detected in NFP-loaded PLA−PEG micelles + H2O2 treated cells (Figure 4A.b), indicating that the NFP-loaded PLA−PEG micelles could successfully maintain the mitochondrial membrane potential under oxidative stress and thus protect the mitochondrial function and cell metabolism. The cataract prevention was also studied in vitro. The lenses of 6-week SD rats were extracted and divided into four groups. Then, the lenses were treated with various culture conditions. After incubation for 24 h, the transparency of lenses was observed under a microscopy. Normal lens was transparent (Figure 4B.a), and the horizontal and vertical lines in paper could be observed clearly through it. Besides, transparent lens could also be noticed in NFP-loaded PLA−PEG micelles + H2O2 treated group (Figure 4B.b). However, the lens treated with H2O2 was completely blurred, indicating that the cataract had formed (Figure 4B.d). This is mainly due to the protein aggregation and precipitation in apoptosis cells that damaged by oxidative stress. Moreover, free NFP could not prevent the cell apoptosis absolutely. In NFP + H2O2 treated lens (Figure 4B.c), only parts of lines in paper could be observed. Results indicated that NFP-loaded PLA−PEG micelles could effectively prevent cell apoptosis and inhibit cataract formation under oxidative stress. The paraffin sections of each group lenses were also studied by hematoxylin-eosin (HE) staining. As shown in Figure 4C.a, the normal lens had a integrate morphology. The outer capsule membrane of normal lens was continuous, and lens epithelial cells arranged orderly. There was a distinct boundary between the cortex and the nucleus. Importantly, the morphology of lens treated with NFP-loaded PLA−PEG micelles was similar to that in normal lens (Figure 4C.b). The result indicated that NFP-loaded PLA−PEG micelles could effectively maintain the structural integrity of lens under oxidative stress, which was more efficient than free NFP (Figure 4C.c). However, the lens treat with H2O2 was damaged seriously by oxidative stress. The outer capsule membrane was detached from cortex, and the lens epithelial cells were scattered. The boundary between cortex and nuclear was blurred (Figure 4C.d). In this study, classic vasodilator NFP was loaded onto a FDA-approved amphiphilic polymer PLA−PEG to form nanosized NFP-loaded PLA−PEG micelles as a new approach to treat oxidative cataract at its early stage. Results indicated that PLA−PEG could significantly improve the solubility and bioavailability of NFP. Moreover, the NFP-loaded PLA−PEG micelles possess the anticataract ability through the effective inhibition of extracellular calcium ions influx when lens was under oxidative stress. Therefore, the NFP-loaded PLA−PEG micelles would have the potential to be used as an eye drop to prevent the formation and progression of oxidative cataract at the early stage. This work would not only supply a new application field for NFP but also provide a new approach in the treatment of cataract.

PEG micelles + H2O2 treated cells, the mean value of the fluorescence intensity was only 3.3 × 105, which was almost the same as that in normal cells (3.1 × 105). Cytochrome c releasing and caspase-3 activation are two significant processes during apoptosis, and the expression of cytochrome c and caspase-3 may help us understanding the cell status in different treated groups. As shown in Figure 2C, H2O2 caused serious damage to the cells, and the expression level of cytochrome c and caspase-3 was increased remarkably, demonstrating that the apoptosis pathway was activated. Moreover, the expression of two proteins in NFP + H2O2 treated group was also elevated. However, cytochrome c and caspase-3 expression levels in NFP-loaded PLA−PEG micelles + H2O2 treated cells was as low as that in the normal cells group, suggesting that the apoptotic pathway mediated by mitochondria has not been activated. The result indicated that the hydrosolubility, stability, and bioavailability of NFP were improved through the encapsulation in PLA−PEG diblock copolymer, and the NFP-loaded PLA−PEG micelles could completely prevent of the calcium influx. The reactive oxygen species (ROS) is mainly generated by mitochondria during respiration, and the ROS level would increase in damaged mitochondria. Therefore, the intracellular ROS level reflects the mitochondrial function and cell status. In this study, DCFH-DA as the intracellular ROS fluorescence probe was used to measure the ROS level in different treated cells. The DCFH-DA was first hydrolyzed into DCFH by intracellular esterase, and then the nonfluorescent DCFH was oxidized by intracellular ROS to generate green fluorescent DCF. The green fluorescence of DCF was detected to determine the intracellular ROS level. As shown in Figure 3A, negligible green fluorescence of DCF could be found in NFP-loaded PLA−PEG micelles + H2O2 treated group (Figure 3A.b). The result was similar to that in normal cells (Figure 3A.a), indicating that NFP-loaded PLA−PEG micelles could effectively maintain the mitochondrial function under oxidation environment. However, a great amount of green fluorescence could be observed in H2O2 treated group (Figure 3A.d), suggesting that mitochondrial function was disorder and metabolism of cells was impaired. In NFP + H2O2 treated cells (Figure 3A.c), the ROS level was significantly higher than that in NFP-loaded PLA−PEG micelles + H2O2 treated cells. It was ascribed to the improved solubility and bioavailability of NFP with the protection of PLA−PEG diblock copolymer.24 Live−dead cell staining assay was performed to measure cytomembrane integrity and cell status (Figure 3B). In normal cells (Figure 3B.a), calcein-AM was endocytosed and hydrolyzed into green fluorescent calcein, while propidium iodide (PI) could not penetrate cytomembrane freely, and the red fluorescence of PI could not be detected. The same result could also be found in NFP-loaded PLA−PEG micelles + H2O2 treated cells (Figure 3B.b), demonstrating that cells were effectively protected from oxidative stress. However, in NFP + H2O2 and H2O2 treated cells, the cytomembrane was damaged. As a result, the calcein-AM could not be successfully hydrolyzed, the red fluorescent PI could accumulate in cell nucleus (Figure 3B.c,d). Mitochondrial membrane potential (△Ψm) plays a vital role in the maintenance of mitochondrial function and cell metabolism. Besides, the depolarization of △Ψm is one of important signals in the early stage of cell apoptosis. Here, the mitochondrial membrane potential of HLECs was measured by JC-1 probe, and the fluorescence of JC-1 was observed E


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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.8b01089.

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Materials and methods, statistical analyses, cytotoxity of PLA−PEG and NFP (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Email: [email protected] (Y.X.S.). *Email: [email protected] (X.Z.Z.). ORCID

Xian-Zheng Zhang: 0000-0001-6242-6005 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (51690152 and 51473126). REFERENCES

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PLAâPEG Micelles Loaded with a Classic Vasodilator for Oxidative

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