Biodegradable PLGA Nanoparticles Restore Lysosomal Acidity and

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Bioengineering

Biodegradable PLGA nanoparticles restore lysosomal acidity and protect neural PC-12 cells against mitochondrial toxicity Jialiu Zeng, Andrew Martin, Xue Han, Orian S. Shirihai, and Mark W. Grinstaff Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.9b02003 • Publication Date (Web): 16 Jul 2019 Downloaded from pubs.acs.org on July 22, 2019

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Industrial & Engineering Chemistry Research

Biodegradable PLGA nanoparticles restore lysosomal acidity and protect neural PC-12 cells against mitochondrial toxicity Jialiu Zeng,†* Andrew Martin,† Xue Han, Orian S. Shirihai and Mark W. Grinstaff* Jialiu Zeng, Andrew Martin, Prof. Xue Han 1.

Department of Biomedical Engineering, Boston University, Boston, MA 02215, United States

Prof. Orian S. Shirihai 2, 3 2.

Division of Endocrinology, Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90045, United States

3.

Department of Medicine, Boston University School of Medicine, Boston, MA 02118, United States

Prof. Mark Grinstaff, 1, 3, 4 1.

Department of Biomedical Engineering, Boston University, Boston, MA 02215, United

states

ACS Paragon Plus Environment

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Industrial & Engineering Chemistry Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Department of Medicine, Boston University School of Medicine, Boston, MA 02118,

United States 4.

Department of Chemistry, Boston University, Boston, MA 02215, United States

†These

authors

have

contributed

equally

to

this

work.

* Co-corresponding authors. E-mails: [email protected] and [email protected] ORCID Zeng, 0000-0001-7802-1432 Martin, 0000-0002-7512-5049 Han, 0000-0003-3896-4609 Shirihai, 0000-0002-9502-2661 Grinstaff, 0000-0002-5453-3668 KEYWORDS PLGA nanoparticles, Lysosomal acidification, Autophagic flux, MPP+, PC-12 cells, Parkinson’s disease

ABSTRACT

Exposure of mitochondrial parkinsonian neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+) to PC-12 cells results in significant cell death, decreases lysosomal acidity, and inhibits autophagic flux. Biodegradable poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) of ≈100 nm diameter localize to the lysosome, degrade, and subsequently release their acidic components to acidify the local lysosomal environment. The performance of PLGA NPs with different lysosomal pH modulating capabilities is investigated in PC-12 cells under MPP+ induced mitochondrial toxicity. PLGA NPs perform in a compositional dependent manner, where NPs with a higher glycolic acid to lactic acid ratio content degrade faster, and yield greater degrees of lysosomal pH modulation as well as autophagic flux modulation in PC-12 cells under MPP+ insult. These results


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show that slight compositional changes of the polymeric NP give rise to differing degrees of lysosomal acidification in PC-12 cells and afford improved cellular degradative activity (142 words).

1. INTRODUCTION

Parkinson’s disease (PD) is the second most common neurodegenerative disorder1 with about 60,000 new cases identified each year (Parkinson’s Foundation). PD results mainly from the death of dopaminergic neurons in the substantia nigra pas compacta (SNc)1,2, due to accumulation of toxic proteins such as alpha synuclein (α-syn) within Lewy bodies and neurites of the nervous system that are unable to be degraded2,3. Being post-mitotic, neurons are unable to dilute damaged organelles or toxic protein aggregates and must clear cellular waste with high efficiency. Recent studies demonstrate that perturbations in the autophagy-lysosome pathway (ALP), which are essential to maintain proper protein and organelle quantity and quality, play a central role in PD pathogenesis3–8. In macro-autophagy, the lysosomal compartment must be maintained at a relatively acidic pH of 4.5 to 5.0 to ensure fusion between lysosomes and autophagosomes9 (Figure 1). Growing evidence indicates that lysosomal acidification is impaired in PD, which subsequently compromises lysosomal enzymes function10,11 and prevents autophagic degradation of toxic proteins. Therefore, targeting lysosomes by modulating their acidity and eventually accelerating autophagic flux represent a new target for therapeutic development. Other pharmacological or genetic enhancement of autophagy, such as rapamycin, are reported to be beneficial in experimental models of PD7,12, however, these therapies are associated with complications including unspecific targeting of downstream cellular processes13. Thus, targeting and restoring autophagy is a novel and promising therapeutic strategy for PD. Herein, we present the synthesis


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and characterization of biodegradable poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) that allow for differing degrees of continuous acid release based on their composition. In a 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced model of PD in PC-12 cells14, treatment with NPs restores both lysosomal acidity and autophagic flux. We chose PLGA from among other biodegradable polymers such as polyethylene terephthalate (PET), polybutylene succinate (PBS), or polycaprolactone (PCL) because PLGA degrades at a faster rate in vivo15 than the other polymers, and yield acids with the lowest pKas among these polyesters (3.85 and 3.83 for glycolic acid and lactic acid respectively). Therefore, NPs composed of PLGA may exert the greatest lowering of lysosomal pH upon intake into the cells and subsequent degradation. While 50:50 PLGA NPs have been reported for their ability to modulate lysosomal pH in ATP13A2mutant or depleted cells and glucocerebrosidase (GBA)-mutant cells16, we are the first to study how the degree of lysosomal pH modulation differs based on changes in copolymer composition, namely the lactic acid to glycolic acid (LA:GA) ratio, in PLGA NPs in PC-12 cells. PLGA copolymers demonstrate varying degradation rates as a function of monomer composition17 when formulated as particles18, foams19, and thin films20. Increasing composition of glycolic acid from 0 to 50% correlates with increasing degradation rate and monomer concentration in the aqueous medium18. Here our work levies the effect of polymer degradation to controllably modulate lysosome acidification and restore autophagic flux. PLGA NPs of variable composition and small size (≈100 nm) are uptaken into the cells and localize to lysosomes. Upon contact with the lysosomal environment, the PLGA NPs degrade and release acid to reduce lysosomal pH, restore autophagic flux, and improve cellular function in compromised PC-12 cells under MPP+ insult.


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Figure 1. In diseased cells under MPP+ insult (left), lysosomal pH is elevated and autophagic flux is inhibited. Addition of NPs release acid to lower lysosomal pH (right), thereby restoring fusion with autophagosomes and restoring autophagic flux. Diseased (MPP+ toxicity) Autophagosome

MPP+

MPP+ toxin elevate lysosomal pH

No fusion

Lysosome (pH ~5.5)

LC3II

With NPs

LC3II

p62

Autophagosome

p62

NPs

NPs release acid to lower lysosomal pH

Blocked degradation of contents

Fusion ensues

Lysosome (pH ~4.5)

Degradation of contents

2. EXPERIMENTAL SECTION PLGA nanoparticles and Rho-PLGA NPs synthesis and characterization: 50 mg PLGA Resomer® RG 503 H, molecular weight (MW) 24,000 – 38,000 g/mol, LA/GA 50:50 (Sigma Aldrich), or PLGA Resomer® RG 752 H, MW 4,000 – 15,000 g/mol, LA/GA 75:25 (Sigma Aldrich) or Poly(D,L-lactic acid), MW 20,000 – 30,000 g/mol (Polysciences Inc), was dissolved in 0.5 mL dichloromethane (DCM), and 32 mg sodium dodecyl sulfate (SDS) was added in 1.5 mL of DI water. The organic and aqueous phase were then combined and sonicated for 10 min at 80 W with a 1 s pulse, 2 s delay under argon atmosphere to create an oil-in-water emulsion suspension. After sonication, the system was opened to the air for 2 h to evaporate excess DCM and dialyzed in DI water for 24 h to remove excess surfactants and salts.


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For synthesis of Rho-PLGA NPs, the same procedure was used except for the addition of Rhodamine B 0.1 mg/mL together with the PLGA polymer in the organic phase. For Dynamic Light Scattering (DLS) measurements, 200 µL of the solution was diluted in 2.8 mL of DI water, and a Brookhaven dynamic light scattering instrument was used to obtain the size and zeta potential. Scanning Electron Microscopy: NPs were diluted 1000 times in DI water, and an aliquot were plated on silicon wafers and air dried overnight. The wafers were then affixed to aluminum stubs with copper tape and sputter coated with 5 nm Au/Pd. These samples were then imaged using a Zeiss Supra 55VP field emission scanning electron microscope using a working distance of 6 cm and an accelerating voltage of 3 kV. Cell culture and mitochondrial activity: PC-12 cells were cultured in DMEM media supplemented with 10% heat inactivated horse serum (HIHS), 5% fetal bovine serum (FBS), 1mM glutamine, 50 µM B-mercaptoethanol, 50 units/ml penicillin, and 50 g/mL streptomycin. The toxicity of PLGA NPs was evaluated using an MTS cell metabolic activity (mitochondrial activity) assay (Abcam). PC-12 cells were cultured in a 96-well plate at 15000 cells/well for 1 day, and subsequently the media was exchanged for media containing either no treatment or 0, 50, 250, 500, 1000 or 5000 µg/mL of either PLGA NPs for 24 hours. MPP+ were treated to the cells at 0, 50, 100, 200, 500 and 1000 µM for 24 hours. The background absorbance was corrected, and the mitochondrial activity was quantified relative to the no treatment control. Flow cytometry: PC-12 cells were cultured at 2 × 105 cells/well in a 96-wellplate overnight. Rhodamine labelled 50:50 PLGA, 75:25 PLGA and PLA NPs were then added at 100μg/mL. Cells were incubated with different NPs for 24 hours. Subsequently, the cells were washed with PBS, and incubated with CalceinAM (Sigma Aldrich) cell viability stain at 1 nM for 30 minutes. The


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cells were washed with PBS again, and prepared for flow cytometry analysis on Attune NxT flow cytometer. PLGA NPs lysosome co-localization imaging: For co-localization imaging, cells were first incubated with Rho-PLGA-NPs for 24 hours, and LysoTracker blue DND-22 dye (Excitation/Emission wavelength: 373/422 nm) was added according to manufacturer’s protocol for 2 hours. The media was then removed, and cells were incubated with fresh media and imaged immediately using Olympus FV1000 scanning confocal microscope. Lysosomal pH analysis: Lysosomal pH measurements were done based on a protocol described in detail previously16. In brief, PC-12 cells were removed from the incubator and washed with PBS solution, and incubated with 5 µM LysoSensor Yellow/Blue DND-160 for 5 min. The dye exhibits a pH-dependent emission at 440 nm and 535 nm, hence permitting ratiometric assessment of pH changes in lysosomes. LysoSensor dye was removed from plate wells after 3 mins, followed by the addition of either 100 µL cell media or pH calibration buffers. pH calibration buffers were made from 10 µM H+/Na+ ionophore Monensin and 20 µM H+/K+ ionophore Nigericin in MES buffer at pH 4.0–6.0, and lysosomal pH values were calibrated in each plate at the same time as experimental levels. Fluorescence was measured with Biotek Synergy HT plate reader. Western blot: Samples were prepared as described previously21. They were loaded in 4-12% polyacrylamide gel (Invitrogen) and transferred onto a polyvinylidene difluoride membrane (Invitrogen) using a wet transfer tank. LC3A/B (Cell Signaling), GAPDH (Cell Signaling), and p62 (Cell Signaling) antibodies were used according to the manufacturer’s instructions. Statistical Analysis: When evaluating all results, predetermined and appropriate statistical methods will be used to determine significance. The mitochondrial activity was quantified relative to the no treatment control, after correcting for background absorbance. PBS buffer pH changes,


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cellular lysosomal pH and autophagic flux protein expression levels were expressed as mean ± S.D. Three independent experiments are conducted (n = 3) for mitochondrial activity, lysosomal pH and autophagic flux protein expression levels. Statistical analysis was performed using GraphPad Prism software; and unpaired t test was used to validate statistical differences between two conditions and p values < 0.05 are considered statistically significant. 3. RESULTS AND DISCUSSION 3.1 Synthesis and characterization of PLGA NPs To evaluate the potential differences between NP formulation and their effect on acidifying diseased lysosomes, we synthesized NPs with three different lactic acid (LA) to glycolic acid (GA) ratios—100:0, 75:25, and 50:50 (LA:GA)— and evaluated their ability to first acidify buffered solutions over time. All of the synthesized NPs exhibit spherical morphology as characterized by scanning electron microscopy (SEM) (Figure 2A), with diameters between 100 – 110 nm. Zeta potential measurements reveal values between -35 and -30 mV for all of the NPs (Figure 2B). Since PLGA polymer degradation rate is related to the amount of glycolic acid in the polymer18, we hypothesized that increasing glycolic acid content, from 0 to 50%, would result in faster degradation and thus greater acidification of the environment. To demonstrate this, we incubated all NP formulations at 10 mg/mL in 20 mM phosphate buffer saline (PBS), at different starting pH, and measured the changes in pH over a week. The lysosomal buffering capacity is ≈20 mM22, and a pH 6.0 environment is reported in other disease models to mimic the dysfunctional lysosomal environment21,23. 50:50 PLGA NPs acidify the solution at a faster rate than 75:25 PLGA NPs and PLA NPs. There was no observable acidification among all NPs during the first two days of incubation. From the second to the third day of incubation, the pH of the 50:50 PLGA NPs treated solution dropped from pH 6.0 to pH 5.7, whereas for 75:25 PLGA NPs the pH of the buffer only


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decreased from pH 6.0 to pH 5.9. There was no change in pH for the PLA NPs (Figure 2C). The 50:50 PLGA and 75:25 PLGA NPs experienced a steady rate of acidification from day 4 to day 7, lowering the pH to 5.5 and 5.7 respectively, while PLA NPs maintained the pH at 6.0. Based on these measurements, we estimated a 2% degradation of the 50:50 PLGA NPs (Supplementary information Equation S1). There were no significant pH changes observed when the NPs were incubated in pH 7.4 (Figure 2D), demonstrating that significant acid is only released on this time scale in a slightly acidic environment (i.e., pH 6.0). Figure 2. NPs morphology and size characterization, and acidification capability of 20 mM pH 6.0 and pH 7.4 buffer. A) SEM micrographs of different NPs show that they have spherical morphology and size around 100 – 110 nm. B) Dynamic light scattering and zeta potential characterizations of NPs. C – D) NPs acidification in 20 mM PBS pH 6.0 and pH 7.4 buffer respectively. (Data=mean ± SD, n = 3 independent experiments). Figure 2 (updated) A

B

PLA

75:25 PLGA

200 nm

200 nm

50:50 PLGA

200 nm

Type of NPs

Size (nm)

Zeta potential (mV)

PLA

108.2 ± 5.7

-30.8 ± 1.1

75:25 PLGA

98.7 ± 6.2

-31.4 ± 1.2

50:50 PLGA

101.5 ± 4.5

-30.2 ± 2.1


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C

D

6.0

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PLA 75:25 PLGA 50:50 PLGA

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Figure 2

pH

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6.6 6.2 6.0

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2 3 4 Time (Days)

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PLA 75:25 PLGA 50:50 PLGA

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3.2 Localization of PLGA NPs and mitochondrial activity in PC-12 cells The NPs were next evaluated for their ability to localize to lysosomes. Rhodamine labelled NPs were incubated with PC-12 cells for 24 hours and then imaged to evaluate colocalization. The 24 hours timepoint was chosen because it has been reported that nearly 100% of cells have taken up PLGA NPs by 24 hours24–26. All three types of NPs readily localized to the lysosomes (Figure 3A – C). The mechanism of PLGA NPs cellular uptake has been previously reported to be through caveolin and clathrin-dependent endocytosis, and Rab34-mediated macropinocytosis27. Flow cytometry showed that cellular uptake of the different NPs was comparable (Supplementary information Figure S1). To determine the effect of NPs on PC-12 cells’ mitochondrial activity, we used an MTS assay to examine and compare the mitochondrial metabolic activity of the cells at various NPs concentrations (Figure 3D – E). 50:50 PLGA and 75:25 PLGA NPs did not exhibit significant reduction in mitochondrial activity up to 500 µg/mL, while PLA exhibited reduction in mitochondrial activity at 500 µg/mL. To minimize toxicity associated with NPs dosing, a 100 µg/mL concentration for all the NPs was chosen for the remaining assays. Figure 3. Cellular uptake of PLGA NPs and mitochondrial toxicity of PLGA NPs in PC-12 cells. A – C) Rhodamine labelled NPs co-localize with LysoTracker blue dye in the lysosomes (Scale bar = 25 µm). D – E) Mitochondria activity analysis of NPs using MTS assay. The mitochondrial


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activity of untreated control cells was assigned a value of 100%. 3E is a close up of the 3D from 0 to 1000 µg/mL (Data=mean ± SD, n = 3 independent experiments). A Rho-PLA NPs

LysoTracker blue

DIC

Merged

B

Rho-75:25 PLGA NPs

LysoTracker blue

DIC

Merged

Rho-50:50 PLGA NPs

LysoTracker blue

DIC

Merged

Figure 3

D

E 100 80 60 PLA 75:25 PLGA 50:50 PLGA

40 20 0

0

1000 2000 3000 4000 5000 Concentration (g/mL)

Mitochondrial activity (%)

C


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100 80 60 PLA 75:25 PLGA 50:50 PLGA

40 20 0

0

200 400 600 800 1000 1200 Concentration (g/mL)

3.3 PLGA NPs rescue MPP+ induced mitochondrial dysfunction The PLGA NPs were next assessed for their efficacy in rescuing mitochondrial activity caused by the mitochondrial neurotoxin MPP+. MPP+ reproduces several PD related cellular changes, such as mitochondrial complex I inhibition, BAX activation, and increased reactive oxygen species


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(ROS) production16. Co-treatment of NPs and MPP+ (50 µM, 100 µM and 1000 µM) for 24 h to PC-12 cells successfully rescued MPP+ induced mitochondrial dysfunction (Figure 4). Compared to 75:25 PLGA NPs and PLA NPs, 50:50 PLGA NPs exhibited higher degrees of mitochondrial activity rescue (Figure 4A – C). This result is due to the higher rate of degradation of 50:50 PLGA NPs, compared to 75:25 PLGA NPs and PLA NPs. However, when the MPP+ concentration was increased 10-fold higher to 1000 µM, the NPs were not able to fully restore mitochondrial activity, unlike MPP+ at 50 µM and 100 µM (Figure 4C). This finding is likely because of insufficient acid release to rescue the significant insult generated by MPP+, due to the slow degradation of PLGA polymers. PLA NPs, with the slowest rate of degradation, were not able to rescue any activity. We also assessed the efficacy of NPs as a neuro-protectant when they were pre-incubated with the PC12 cells for 16 h before MPP+ insult for 24 h (Supplementary information Figure S2A – C). The NPs protected cells against MPP+ induced mitochondrial dysfunction. Figure 4. PLGA NPs rescue MPP+ induced mitochondrial dysfunction. A – C) Co-treatment of PLGA NPs with MPP+ at 50 µM, 100 µM and 1000 µM for 24 h reduce MPP+ resulted mitochondrial dysfunction. (Data=mean ± SD, n = 3 independent experiments, *=p < 0.05, ***=p< 0.001, n.s. = not statistically significant)

*

n.s.

100 80 60 40 20

0 trol µM A NPs A NPs A NPs Con P+ 50 PL LG LG MP M with 5:25 P 0:50 P 7 ith 5 50 µ with w + P MP + 50 µM+ 50 µM P P MP MP

B 120 100

***

*

**

***

80 60 40 20

0 Ps Ps Ps trol µM Con + 100 PLA N LGA N LGA N th :25 P :50 P P i P w M 75 50 µM 100 M with M with P+ MP + 100 µ + 100 µ P P MP MP

C 120 Mitochondrial activity (%)

120

***

n.s.


A 140 Mitochondrial activity (%)

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***

100 80

***

***

n.s.

60 40 20

0 trol µM A NPs A NPs A NPs Con 1000 L G LG + ith P 5 PL 0P P MP 0 µM w h 75:2 h 50:5 t t 100 µM wi µM wi P+ MP + 1000 + 1000 P P MP MP

3.4 PLGA NPs restore lysosomal acidification after lysosomal inhibition Figure 4 (updated) ACS Paragon Plus Environment

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We further investigated if the restoration of MPP+ induced reduction in mitochondrial activity arose from the lysosomal acidity modulation capabilities of the NPs. Treatment of cells with either 50 µM, 100 µM or 1000 µM MPP+ alkalinizes lysosomes, elevating lysosomal pH from the basal value of 4.5 to above 5.0 (Figure 5A – C). When the cells were co-incubated with MPP+ and NPs for 24 h, lysosomal pH was restored to different degrees. Under 50 µM and 100 µM MPP+ insult, NPs restored lysosomal pH levels. However, at 1000 µM MPP+ insult, NPs displayed reduced capability to restore lysosomal pH. PLA NPs did not induce significant lysosomal pH changes, while 75:25 PLGA and 50:50 PLGA NPs were able to partially recover lysosomal pH from 5.2 to 5.0. This observation agrees with the pH changes profiled in Figure 2, where 50:50 PLGA NPs exhibited the fastest degradation rate, releasing more acid and restoring lysosomal pH more significantly than either 75:25 PLGA or PLA NPs. However, the overall degradation rate and acid release amount was insufficient to rescue MPP+ induced lysosome and mitochondrial dysfunction at higher levels of MPP+ insult. Figure 5. Co-incubation of PLGA NPs with MPP+ at 50 µM, 100 µM or 1000 µM modulate lysosomal pH. (Data=mean ± SD, n = 3 independent experiments, ***=p< 0.001, n.s. = not statistically significant)

5.0

***

*

*

***

B

4.6 4.4 4.2 4.0

trol µM A NPs A NPs A NPs Con P+ 50 L G G P ith P :25 PL :50 PL w M 0 5 M 5 7 µ h h 0 5 wit wit P+ MP + 50 µM + 50 µM P P P P M M

Figure 5 (updated)

Lysosomal pH

4.8

***

n.s.

*** **

***

C

5.0

5.2

4.8

5.0

4.6 4.4 4.2

Lysosomal pH

A

Lysosomal pH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


n.s.

***

n.s.

4.8 4.6 4.4 4.2

4.0

4.0 Ps Ps Ps trol trol µM µM A NPs A NPs A NPs Con 1000 PLA N LGA N LGA N Con + 100 L G G P L L P P h P P h + t t P i i 5 0 P MP µM w 75:25 50:50 MP 0 µM w h 75:2 h 50:5 t t with with 100 100 µM wi µM wi + P+ 0 µM 0 µM P P M + 10 10 MP + 1000 + 1000 P+ P P P MP MP MP MP


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3.5 PLGA NPs restore autophagic flux inhibition due to lysosomal inhibition To determine if the lysosomal pH restoration was able to improve autophagic degradative function, autophagic flux was measured in PC-12 cells co-incubated with NPs and MPP+. Two major ALP proteins were quantified for their expression levels - LC3-II, microtubule-associated proteins 1A/1B light chain 3B and the lipidated form of LC3-I, which is found on autophagosome membrane, and p62, a receptor usually sequestered by autophagosomes, using western blot densitometry (Figure 6A – C). PC-12 cells treated with 50, 100 or 1000 µM MPP+ exhibited a significant increase in LC3-II and p62 levels compared to control cells with no treatment, indicating an accumulation of autophagosomes and decreased autophagic degradation. Under MPP+ 50 µM insult, all NPs significantly restored autophagic flux inhibition (Figure 6A, D and G). Under MPP+ 100 µM insult, PLA NPs did not restore autophagic flux, while both 75:25 PLGA and 50:50 PLGA NPs significantly increased clearance or reduction of LC3-II and p62 (Figure 6B, E and H). 50:50 PLGA NPs slightly attenuated LC3-II levels and significantly attenuated p62 levels, indicating a slight recovery of autophagic flux under 1000 µM MPP+ insult (Figure 6C, F and I). This is consistent with 50:50 PLGA NPs demonstrating the most significant restoration of lysosomal pH among all NPs. However, this amount of lysosomal pH attenuation was unable to fully restore autophagic flux under high concentrations of MPP+ insult. Figure 6. Co-incubation of PLGA NPs with 50 µM (A, D, G), 100 µM (B, E, H), or 1000 µM (C, F, I) MPP+ modulate autophagic flux. Untreated control cells LC3-II and p62 expression levels were assigned a value of 1. LC3-II and p62 expression levels for other treatment conditions were normalized to control. Representative western blot images for MPP+ concentration is shown here. (Data=mean ± SD, n = 3 independent experiments, *=p < 0.05, **=p < 0.01, ***=p< 0.001, n.s. = not statistically significant)


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A

Co-incubate 50 µM

B

Co-incubate 100 µM

C

Co-incubate 1000 µM

GAPDH

*

*

*

**

E

3.0 2.5

1.5 1.0 0.5 0.0

G

1.2

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1.0

*

**

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0.8 0.6 0.4

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F

*

2.4

1.5 1.0

0.8 0.4 0.0

n.s.

*

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4.0 3.0 2.0 1.0

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*

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* **

1.6

0.5

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2.0

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Ps Ps Ps trol µM Con P+ 50 PLA N LGA N LGA N MP M with 5:25 P 0:50 P 5 7 µ 50 with with P+ M P + 50 µM + 50 µM P P MP MP

*

Ps Ps Ps trol µM Con + 100 PLA N LGA N LGA N P P h P MP µM wit 75:25 50:50 100 M with M with P+ M P + 100 µ + 100 µ P P MP MP

LC3II/GAPDH

Ps Ps Ps trol µM Con P+ 50 PLA N LGA N LGA N MP M with 5:25 P 0:50 P 7 5 50 µ with with P+ MP + 50 µM+ 50 µM P P P P M M

n.s.

p62/GAPDH

p62/GAPDH

2.0

p62/GAPDH

D

Ps Ps Ps trol µM Con + 100 PLA N LGA N LGA N P P h P MP µM wit 75:25 50:50 100 M with M with P+ MP + 100 µ + 100 µ P P MP MP

Ps Ps Ps trol µM Con 1000 PLA N LGA N LGA N ith 5P 0P P+ MP 0 µM w h 75:2 h 50:5 100 µM wit µM wit P+ MP + 1000 + 1000 P P MP MP

I LC3II/GAPDH

p62 LC3I LC3II

LC3II/GAPDH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


n.s.

2.0 1.5

**

1.0 0.5 0.0

Ps Ps Ps trol µM Con 1000 PLA N LGA N LGA N ith 5P 0P P+ M P 0 µ M w h 7 5 :2 h 5 0 :5 100 µM wit µM wit P+ M P + 1000 + 1000 P P MP MP

4. CONCLUSION Lysosomal impairment is increasingly regarded as a major contributor to the pathogenesis of neurodegenerative diseases, including idiopathic and genetic forms of PD. However, none of the strategies in targeting ALPs directly target the lysosome, but instead enhance the entire autophagy machinery. Here we show that PLGA NPs internalize into PC-12 cells and are readily trafficked to lysosomes. Furthermore, we reveal that PLGA NPs re-acidify defective lysosomes caused by mitochondrial toxin MPP+, and rescue MPP+ induced mitochondrial toxicity in PC-12 cells. However, the re-acidification and rescue of mitochondrial activity was only partial in PC-12 cells under high concentrations of MPP+ insult at 1000 µM. Moreover, this restoration effect is also dependent on NP composition, with greater glycolic acid content in the NP affording a more potent response. Our data, and previous work18, show that increasing polymeric glycolic acid content up to 50% affords more rapid NP degradation and generates more acid in solution. The above findings also connect the NP compositional dependence on acidification with NP ability to acidify


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lysosomes and restore autophagic flux, resulting in restoration of mitochondrial activity. The NPs also rely on a slightly acidic environment, like that of an inhibited lysosome, to degrade and enact their effect (Figure 2 C – D) upon a pH trigger. Studies have shown that when Bafilomycin A1, a lysosomal V-ATPase inhibitor, is added to the PC-12 cells16, 50:50 PLGA NPs have reduced efficacy in restoring lysosomal pH, suggesting that a partially functional V-ATPase is required for the NPs to exert their effect. Additionally, our work adds to other current approaches in rescuing MPP+ induced PC-12 mitochondrial toxicity, such as the use of Salidroside28 and Resolvin D129 to reduce ROS and inflammatory cytokines levels, or Fisetin to potentially inhibit several apoptotic and inflammatory pathways30. From a design perspective, the possibility to fine tune the acidifying capability of these nanoparticles could enable investigation of diseases that have implicated lysosomal pH dysfunction pathology, but require differential release times or dosage regimes. From a clinical perspective, nanoparticles capable of acidifying lysosomes could serve as potential nanotherapeutics for pathologies associated with lysosomal impairment and are worthy of further evaluation.

Supporting Information. Equation S1. Equations used to determine the amount of monomer needed to acidity PBS buffer. Figure S1. Uptake of rhodamine-labeled 50:50 PLGA, 75:25 PLGA and PLA NPs in PC-12 cells was monitored using flow cytometry after 24 hours of incubation. Cellular uptake was comparable among different NPs. Figure S2. PLGA NPs pre-treated for 16 hours before MPP+ at (A) 50 µM, (B) 100 µM and (C) 1000 µM insult rescue PC-12 mitochondrial activity. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Authors *Jialiu Zeng [email protected] and Mark W. Grinstaff [email protected]


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Author Contributions JLZ designed, conducted and analyzed all experiments. AM contributed to PLGA NPs pH measurements, calculations and Rho-PLGA NPs lysosomal co-localization imaging, and performed some data analysis. JLZ and MWG wrote the manuscript. AM, OSS and XH edited and provided comments to the manuscript. All authors have given approval to the final version of the manuscript. Acknowledgements This contribution was identified by Dr. André Costa (University of Minho) as the Best Presentation in the PMSE session of the 2018 ACS Fall National Meeting in Boston. The authors would also like to thank Eric Bressler for his help on the flow cytometry. Funding Sources This work was funded by National Institute of Health R21 grant NIH AG063373, BU Nanotechnology center at Boston University (JLZ and AM), and T32 grant Translational Research in Biomaterials NIH T32EB00635931 (AM). Conflict of Interest All authors declare no conflict of interest. ABBREVIATIONS ALP, autophagosome – lysosome pathway; MPP+, 1-methyl-4-phenylpyridinium ion; NP, Nanoparticle; PD, Parkinson’s disease; PLGA, poly (lactic-co-glycolic) acid. REFERENCES (1)

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Table of contents MPP+ PLA 50:50 PLGA 75:25 PLGA Autophagosome Autophagosome Autophagosome Autophagosome LC3II p62 NPs

Fusion ensues

No fusion Lysosome (pH ~5.5)

pH ~5.0

No degradation

Fusion ensues

Fusion ensues pH ~4.8

Degradation ensues

pH ~4.5

Degradation ensues

Degradation ensues


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Biodegradable PLGA Nanoparticles Restore Lysosomal Acidity and

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