Preparation and Evaluation of Onjisaponin B-Loaded Liposomes for Drug Delivery to Enhance Mitochondrial Function and Rescue Parkinson’s Disease by Activating Mitophagy

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Introduction
Parkinson's disease (PD) is the second most common neurodegenerative disorder that aficts millions of people worldwide [1].α-synuclein accumulation and Lewy body formation are the pathological hallmarks of PD and eventually cause severe dopamine depletion in the substantia nigra pars compact (SNpc) as well as motor defects, such as bradykinesia and motor rigidity [2].To date, there is no cure for PD, and current therapy is limited to supportive care that partially relives the symptoms [3].
Mitochondria are considered as the "powerhouse" of the cell, and their main function is to generate adenosine 5′ triphosphate (ATP) at the mitochondrial electron transport chain (ETC) [4].Mitochondria also mediate calcium homeostasis, oxidative stress response, and programmed cell death [5].Although the precise mechanism of PD pathogenesis remains elusive, mitochondrial dysfunction has been identifed to have a vital role in the neurodegenerative process of PD [6].Previous evidence suggests that high levels of mitochondrial DNA deletion occurred in the substantia nigra neurons from PD patients [7].In addition, mitochondrial ETC inhibitors such as trichloroethylene and 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine-(MPTP-) induced parkinsonism in humans and experimental animals [5,8].
Autophagy is a highly conserved cellular recycling process that serves to deliver cytoplasmic constituents to lysosomes for degradation, and plays important roles in cell survival and maintenance [9].Te selective degradation of mitochondria by autophagy is termed as mitophagy, which eliminates dysfunctional mitochondria and prevents ATP defciency and ROS accumulation resulting in oxidative stress and cell death [10].PINK1 is a serine/threonine kinase localized at mitochondria, and Parkin is an E3 ubiquitin ligase resides in the cytosol [11].Te PINK1/Parkinmediated mitophagy is the most studied mitophagy pathway.PINK1 detects damaged mitochondria and then recruits Parkin to ubiquitinate the dysfunctional mitochondria for their degradation by autophagy [12].Previous studies have provided clear evidence to support the involvement of the PINK1/Parkin pathway of mitophagy in the pathogenesis and treatment of PD [13,14].
Liposomes, fabricated from the self-assembly of phospholipids, have been widely used as a drug delivery system to the brain [15].Depocyt is a cytarabine liposomal injection that is F.D.A. approved for the treatment of neoplastic meningitis through spinal injection [16].Depocyt could treat neoplastic meningitis with the controlled release of Ara-C.Kasenda et al. carried out evaluation of targeted immunoliposomes for the treatment of epidermal growth factor receptor-(EGFR-) positive glioblastoma by clinical trial.Tey found that anti-EGFR immunoliposomes can target EGFR-amplifed glioblastoma [17].As an ideal nanocarriers for the central nervous system, liposomes improve the bioavailability of drugs and minimize systemic efects of drugs by enhancing BBB penetration, targeting a specifc group of cells, and avoiding premature metabolism [18].Onjisaponin B (OB) is an active compound derived from Radix Polygalae, which exerts neuroprotective efects in neurodegenerative disorders, such as AD and PD [19,20].Existing evidence shows that OB successfully ameliorated DA neurons degeneration and motor defcits by enhancing autophagy and decreasing oxidative stress in a mouse model of PD [19,21].OB was also demonstrated to promote the clearance of α-synuclein mutants (a central component to the pathogenesis of PD) in PC12 cells via autophagy activation [22].However, the therapeutic efect of OB-loaded liposomes has not been studied.
Liposomes as drug carriers have the following three main advantages: (1) simplifed manufacturing method, (2) ability to encapsulate a wide range of drugs and molecules and are not limited by other physicochemical properties such as hydrophobicity and charge, and (3) biocompatibility.In the aqueous environment, the hydrophobic tail forms a spherical structure composed of an aqueous core, which is surrounded by a lipophilic bilayer membrane.Liposomes can be controlled by changing the lipid composition because they are biocompatible and biodegradable.In addition, liposomes can be subjected to various modifcations to enhance their efcacy as drug delivery carriers.
To address this issue, the present study explored the efects of lip OB in cell and mouse models of PD.We found that lip OB rescued motor defects, dopaminergic neurons degeneration in vivo, and improved cell survival in vitro.Furthermore, these efects are related to the restoration of mitochondria dysfunction by activating the PINK1/Parkin signaling of mitophagy.

Lip OB Rescues MPTP-Induced Motor Defcits and Dopaminergic Neurons Degeneration
In Vivo.MPTP is a neurotoxin that can induce PD symptoms in humans and rodent animals by metabolizing into 1-methyl-4-phenylpyridinium (MPP + ) that enters dopaminergic neurons, where it inhibits respiratory chain complexes [23].To evaluate the protective efects of lip OB on PD, an MPTP-induced PD mouse model was employed.Following administration with free or lip OB, the latency to fall (MPTP: 158.9 ± 21.12 s, MPTP + free OB: 183.0 ± 18.79 s, MPTP + lip OB: 212.Next, we examined lip OB-induced efects on dopaminergic degeneration in the SNpc (Figure 1(d)).As expected, TH-positive cells in the MPTP group were greatly reduced as compared to those in the controls.However, free or lip OB treatment greatly inhibited the death of TH-positive neurons (MPTP: 4522 ± 1181, MPTP + free OB: 7553 ± 447.2, MPTP + lip OB: 8397 ± 1024, Control: 11107 ± 1530).It is noteworthy, that lip OB showed better efects than free OB on mitigating MPTP-induced behavioral dysfunction and dopaminergic neurons loss.Collectively, these results suggest that lip OB could ameliorate PD-related motor impairments and dopaminergic neurons degeneration.

Lip OB Remedies MPTP-Induced Mitochondrial Dysfunction In Vivo.
Mitochondrial dysfunction is tightly linked to PD pathogenesis [24].Mitochondrial Complex I is the largest multimeric enzyme complex of the mitochondrial respiratory chain, the inhibition of which leads to ATP depletion and ROS generation responsible for oxidative stress [25].Complex I defciency has been reported in PD patients [24,26].To explore the role of OB in mitochondrial function, we detected the levels of Complex I, ATP, and oxidative stress.As shown in Figure 2, MPTP treatment reduced the activities of Complex I and ATP and elevated the generation of MDA and SOD.Nonetheless, the aforementioned efects were all successfully reversed by lip OB administration.In contrast, free OB only signifcantly rescued the SOD levels.Tese results indicated that lip OB restores MPTP-induced mitochondria dysfunction.

Lip OB Induces Mitophagy by Activating PINK1/Parkin
Pathway.Mitophagy is a selective form of autophagy, which controls mitochondrial quality by removing damaged mitochondria and is involved in the pathology of Parkinson's disease [13,27].Te PINK1/Parkin pathway is reported to play an essential role in triggering mitophagy [28].We thus investigated whether OB-induced improvement in mitochondria function is associated with PINK1/Parkin-  Journal of Clinical Pharmacy and Terapeutics dependent mitophagy.During autophagy, ATG5 and LC3-I to LC3-II conversion are involved in autophagosome formation [29].LAMP2A locates in the lysosomal membrane and is responsible for the protein translocation from the cytosol to the lysosomal lumen [30].P62 is an autophagosome cargo protein that is critical for the clearance of ubiquitinated proteins [30].In the western blotting, MPTPinduced a notable decrease in PINK1 and Parkin expression, accompanied by a signifcant reduction in ATG5 expression, LC3-II/LC3-I ratio and LAMP2A levels, and an increase in P62 expression (Figure 3).Interestingly, these efects were all reversed after treatment with lip OB, while free OB only rescued the protein levels of ATG5 and P62.Together, these data suggest that lip OB activates PINK1/Parkin-mediated mitophagy in the PD mouse model.

Lip OB Improves Cell Survival and Mitochondrial Function in MPP + -Treated PC12 Cells through PINK1/Parkin
Pathway of Mitophagy.To explore the benefcial efects of lip OB on Parkinson's disease in vitro, PC12 cells were pretreated with lip OB for 6 h, followed by exposure to MPP + for 24 h.Given that the maximum concentration that had no efect on decreasing cell viability was 0.  We next tested whether PINK1/Parkin-mediated mitophagy was involved in lip OB-generated benefcial effects in Parkinson's disease in vitro.In line with in vivo results, lip OB successfully blocked MPP + -induced increase in P62 expression and a decrease in PINK1, Parkin, ATG5, and LAMP2A levels, as well as LC3-II/LC3-I ratio (Figures 6(a) and 6(b)).Te GFP-LC3-mitochondria colocalization showed that lip OB attenuated MPP + -induced loss in the colocalization of GFP-LC3 and mitochondria (Figure 6(c)).
Overall, these fndings suggested that the damaging efects of MPP + on neuronal survival and mitochondrial function were well restored with lip OB administration by afecting the PINK1/Parkin pathway of mitophagy.

Silencing PINK1 Abrogated the Protective Efects of Lip OB on MPP + -Treated PC12 Cells.
To investigate whether the PINK1 pathway was required in the protective efect of lip OB against Parkinson's disease, PINK1 siRNA knockdown was performed in PC12 cells.As illustrated in Figures 7(a

Discussion
PD is a common neurodegenerative disorder that is clinically characterized by tremor, gait rigidity, and hypokinesia [31].Te morbidity of PD presents signifcant healthcare, social, and economic issues [32].However, there is no available treatment that can halt the progression of the disease.In the present study, we investigated the therapeutic efect of OB-loaded liposomes on PD.We found that lip OB signifcantly reduced the motor impairment and dopaminergic neuron in PD models.In addition, enhancement of mitochondrial function was observed following lip OB treatment, which was associated with PINK1/Parkin signaling-mediated mitophagy.OB is a major component from the herbal medicine Radix Polygalae.OB possesses strong biological activities and has displayed therapeutic efects in several disease models, including PD [21,33,34].In our work, OB rescued MPTP-induced motor dysfunction and dopaminergic neuron death in a mouse model of PD, confrming the protective role of OB in PD.Furthermore, lip OB displayed a better efect on ameliorating Parkinson-like syndrome relative to free OB, supporting the strength of liposomes served as a drug delivery system to the brain.
Mitochondria are the main sites of biological energy generation in eukaryotes and regulate a wide variety of cellular functions, including ATP production, calcium bufering, ROS production, and scavenging [35].Mounting evidence suggests that mitochondria play an important role in PD [36,37].For example, mitochondrial respiratory chain defciency such as reduced Complex I activities was seen in the skeletal muscle, platelets, and substantia nigra of patients with PD [38].Postmortem studies have shown high levels of oxidation of lipids and proteins in the substantia nigra of PD brains [39].Andrographolide restored mitochondrial functions by inhibiting mitochondrial excessive division, which ultimately mitigated Parkinsonism in MPTP-PD mice [40].Consistent with these studies, we found that MPTP or MPP + modeling impaired mitochondrial respiration and mitochondrial membrane potential and aggravated ROS accumulation and oxidative stress response, suggesting the involvement of mitochondrial dysfunction in PD.While treatment with lip OB attenuated PD symptoms by enhancing mitochondrial functions, it highlighted the critical role of mitochondria restoration in the treatment of PD.
Mitochondria selective autophagy, termed as mitophagy, is a mechanism of elimination of mitochondria via autophagy, and mediates mitochondrial quality control and homeostasis [41].In mammalian cells, the mitochondrial kinase PINK1 and the E3 ubiquitin ligase Parkin act in coordination in monitoring mitochondrial functional state and tags damaged mitochondria for autophagic clearance [42].Te alternation of PINK1/Parkin-mediated mitophagy in PD has been documented in previous studies [12,43].Mutations in the PINK1 and Parkin result in autosomal recessive and juvenile parkinsonism [44,45].δ-opioid receptor (DOR) activation rescued PD-related mitochondrial damage by driving the PINK1/Parkin pathway of mitophagy.While knockdown of PINK1 aggravated mitochondrial dysfunction and compromised DOR-induced cytoprotection against MPP + [46].Huang et al. found that vasicinone suppressed ROS production, improved mitochondrial membrane potential, and prevented paraquat-induced PD through enhancing mitophagy; however, inhibition of mitophagy abolished the ameliorating efect of vasicinone in PD [47].In our study, the PINK1/Parkin pathway of mitophagy was suppressed in the cell and mouse models of PD and was successfully activated following lip OB treatment.Furthermore, PINK1 siRNA knockdown blocked lip OB-generated therapeutic efects in PD.Tese fndings supported the importance of PINK1/Parkin-mediated mitophagy in the etiology and treatment of PD.
In conclusion, this study demonstrated that lip OB plays a protective role in PD by enhancing mitochondria function via PINK1/Parkin-mediated mitophagy.Our study shows the potential of lip OB in treating PD.

Preparation and Characterization of OB-Loaded
Liposomes.OB-loaded PEG/cyclic Arg-Gly-Asp (cRGD) dual-modifed liposomes were prepared using the thin-flm dispersion method [48].Briefy, SPC, cholesterol, PEG-DSPE, and SH-PEG-DSPE were dissolved at a molar ratio of 80 : 15 : 3:2 in chloroform.Tis mixture was rotated in a rotary evaporator at 37 ± 5 °C for 10 min.Te thin flm was dissolved in chloroform and mixed with a 120 mmoL/L calcium acetate solution (pH 7.3).Liposomes were sonicated at 300 W, 5 min in an ice bath, and then fltered through a 220 nM pore size polycarbonate membrane.Liposomes preformed in calcium acetate solution (inside pH 7.3) were rinsed twice in 120 mmoL/L sodium sulfate solution (outside pH 5.9) by ultracentrifugation at 100 000 × g and 4 °C for 60 min.Afterwards, onjisaponin B (>98% purity; MUST Bio-technology) in 120 mmoL/L sodium sulfate solution was added.Liposomes were then ultracentrifugated at 100 000 × g and 4 °C for 60 min.Finally, the liposomes were incubated with the Mal-cRGDyk peptide at an equimolar amount of SH-PEG-DSPE at 37 °C for 2 h.
Te morphology of the prepared liposomes was observed under a transmission electron microscopy (HT7700, HITACHI; Figure S1A).Te size distribution and zeta potential of liposomes were determined using a Nano ZS90 Zetasizer (Figures S1B and S1C).
To determine the body biodistribution profle of lip OB, C57BL/6J mice were injected with MPTP (30 mg/kg) twice a week for 3 weeks.At 6 h post the last MPTP treatment, DiR-labeled liposomes including Lip, Lip-PEG, Lip-cRGD, and Lip-PEG-cRGD were given to mice by intravenous injection.At 12 h postliposome administration, the images of the mice were derived using the fuorescence imaging system IVIS Lumina II (IVIS; Figure S1D).Hereafter, mice were euthanized, and the brains were harvested and imaged using the IVIS.

Animals
Experiments.C57BL/6 mice at 3 months of age were purchased from Shanghai Sipper-BK Laboratory Animal Co. Ltd (Shanghai, China).Te animals were grouphoused and maintained under a 12-hour light/dark cycle with free access to food and water.Te animals were managed according to procedures approved by Jiangsu University.
After a week of acclimatization, mice were divided into four groups at random (n � 9 per group): control, MPTP, MPTP + free OB, and MPTP + lip OB groups.MPTP (30 mg/ kg/day; Sigma) was given by intraperitoneal (i.p.) injection twice a week for 3 weeks.At 6 h post the last MPTP treatment, free and lip OB were administered (i.v.) at a dose of 5 mg/kg.Te control group received i.v. and i.p. injections of the same dose of normal saline.Te MPTP group received i.p. injection of MPTP and i.v.administration of the same dose of normal saline.Twelve hours after OB treatment, the rotarod test and pole test were performed subsequently.Afterwards, mice were euthanized by cervical dislocation for tissues sampling.

4.3.
Cell Culture and Treatment.Te PC12 cell line was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's modifed Eagle medium containing 10% fetal bovine serum (FBS; Gibco) and 1% antibiotic at 37 °C in a humidifed atmosphere containing 5% CO 2 .Cells were treated with 0.2-0.8μM of free or lip OB for 6 h, followed by exposure to 5 μM MPP + (Sigma) for 24 h.For gene knockdown, the cells were transfected with PINK1 siRNA prior to OB administration as described below.Te MPP + concentration and OB treatment time window were determined by the MTT and western blotting assays, respectively (Figure S2).

RNA Interference of PINK1.
To inhibit of PINK1 expression, PC12 cells seeded in 6-well plates were transfected with specifc PINK1 siRNA for 12 h using the Lipofectamine 3000 according to the manufacturer's instructions.Te transfection efciency was evaluated by the western blot analysis (Figure S3).

Rotarod Test.
Motor balance and coordination were evaluated using a rotarod apparatus as previously described [49].Mice underwent two trails (6 min per trail, with an accelerated speed of 12-20 rpm) per day for 2 consecutive days prior to the assessment.Ten, the animals were tested for 5 min (constant 20 rpm).Te latency to fall of the rotating rod was recorded as latency time.A decreased latency time is associated with a motor defcit.4.6.Pole Test.Te pole test was conducted to measure bradykinesia as reported previously [49].Te apparatus consisted of a rough-surfaced wooden pole (50 cm in height, 1 cm in diameter) with a ball placed on top.Te base of the pole was covered with bedding to protect mice from injury.Mice were positioned upward at the top of the pole.Te time required for the mice to completely turn downward and reach the bottom was recorded.All mice were pretrained with the pole three times prior to the assessment.4.7.Immunohistochemical Analysis.Te technique we used was performed as reported previously [50].Sections were washed in PBS, permeabilized in 0.5% Triton X-100 for 10 min, followed by incubation in 3% H 2 O 2 for 20 min.After that, brain slices were blocked with a 5% bovine serum albumin (BSA) solution for 1 h and incubated with an antibody against TH overnight at 4 °C.Te following day, sections were incubated with the corresponding secondary antibodies at room temperature for 2 h.Ten, the slices were stained with 3,3′-diamino-benzidine (DAB), mounted on glass slides, and coverslipped.Digital images of TH neurons in SNpc were captured using an Olympus BX52 microscope.

Mitochondrial Membrane Potential (MMP) Assay.
A JC-1 kit (Invitrogen) was used to observe the mitochondrial potential.Cells were incubated with a diluted JC-1 reagent (1 : 1000) at 37 °C for 20 min.After rinsing with PBS, cells were observed under a fuorescence microscope.At a high MMP, JC-1 forms aggregates (red fuorescence; 550 nm excitation/600 nm emission) inside mitochondria, whereas in a low MMP state, JC-1 remains as a monomer (green fuorescence; 485 nm excitation/535 nm emission).Te ratio of fuorescence red/green fuorescence intensity was quantifed using the ImageJ software (NIH).

Determination of ROS Production.
Te ROS probe dye 2′,7′-dichlorofuorescein diacetate (DCF-DA) was used to determine intracellular ROS generation.Cells were loaded with 10 μM DCF-DA for 30 min and then rinsed in PBS twice.Fluorescent signal detection of cells was performed using a fow cytometry.

LC3B-GFP-Adenoviral
Transfection.LC3B-GFPadenoviral transfection and MitoTracker Red staining were performed to visualize mitophagy as previously documented [52].PC12 cells were transfected with GFP-LC3B adenovirus for 24 h, treated with OB (0.2, 0.4, and 0.8 μM) for 6 h, and then exposed to 5 μM MPP + for another 24 h.After washing in PBS, cells were stained with MitoTracker Red (500 nM, CST) at 37 °C for 30 min to visualize mitochondria.Ten, cells were washed with PBS and fxed in 4% paraformaldehyde for 15 min.Images were captured using a Leica confocal microscope.

Statistical Analysis.
Data were presented as the mean ± standard deviation (SD).Comparison between two groups was analyzed using an unpaired two-tailed t test.Diferences among multiple groups were determined using one-or two-way analysis of variance (ANOVA), followed by a post hoc test (Dunnett's or Sidak's multiple comparisons tests).Statistics were conducted using the GraphPad Prism 8 software.Te criterion of signifcance was set at P < 0.05.cells were pretreated with lip OB for the indicative time and then exposed to 5 μM MPP + for another 24 h.Te LC3-II/ LC3-I ratio and P62 expression were quantifed using the western blotting.Te OB (6 h) group displayed a better efect on activating autophagy (n = 3).Data were expressed as means ± SD. * P < 0.05, * * P < 0.01 compared with the control group.Figure S3.Transfection efciency of PINK1 siRNA.PC12 cells were transfected with PINK1 siRNA for 12 h, and then the western blot assay was performed.PINK1 siRNA caused a signifcant decrease in PINK1 expression.Data were expressed as means ± SD. * * P < 0.01 compared with Scramble siRNA group.(Supplementary Materials)

Figure 1 :
Figure 1: Lip OB reduces motor impairments and dopaminergic neuronal loss in mice exposed to MPTP.(a) Latency to fall in the rotarod test (n � 9).(b, c) Time of turning downward and reach the bottom in the pole test (n � 9).(d) Representative pictures and quantifcation of TH+ neurons in the SNpc of mice exposed to MPTP (n � 3).Scale bar: 100 μm.Data were expressed as means ± SD. * P < 0.05, * * P < 0.01 compared with MPTP group.

Figure 2 :Figure 3 :
Figure2: Lip OB improves mitochondrial dysfunctions in mice exposed to MPTP.Te levels of Complex I, ATP, MDA, and SOD in the SNpc were determined using the commercial kits.Data were expressed as means ± SD (n � 6).* * P < 0.01 compared with the MPTP group.

Figure 4 :
Figure 4: Lip OB inhibits cell survival in MPP + -treated PC12 cells.(a) Screening of lip OB concentrations using the MTT assay (n � 5).(b) PC12 cells were pretreated with lip OB for 6 h, followed by the exposure to MPP + for 24 h.Te cell viability was detected by the MTT assay (n � 5).(c, d) Flow cytometry analysis of the apoptosis ratio of PC12 cells (n � 3).Data were expressed as means ± SD. * P < 0.05, * * P < 0.01 compared with blank or MPP + group.