targeting sequence to induce PLGA nanoparticles for mitochondrial uptake, resulting in improved protective efficacy
against aminoglycosides in common formulations. Nevertheless, absolute protection was absent for the designed
delivery system, probably due to the moderate targeting efficacy [12].
In the present study, we designed and synthesized a series of SS-31 analogues with the aim to improve the
antioxidant efficacy and mitochondrial targeting ability of inducing drug delivery system of the lead compound, which
were known to be important for the treatment of mitochondrial dysfunctional diseases. To gain a systematic insight
into mitochondrial-targeting peptide-mediated delivery, we firstly synthesized a small library of SS-31 analogs. The
aim of optimization was to provide controlled toxicity, favorable antioxidant activity and suitable mitochondrial
delivery. We concluded that the RF-2 peptide indeed facilitates the mitochondrial delivery of PLGA NPs in a non-toxic
manner.
The study was conducted in accordance with the Basic & Clinical Pharmacology &Toxicology policy for
experimental and clinical studies [13]. Peptides in this study were synthesized by the previously described manual
solid-phase procedure, using techniques for Nα-Fmoc-protected or Boc(t-butyloxycarbonyl)-protected amino acids on
Rink amide resin (0.03 mmol scale) and p-methylbenzhydrylamine (MBHA) resin (1% cross-linked, 100~200 mesh,
0.3 mmol/g, Aladdin Industrial Corporation). The NƐ-amino groups of Lys and D-Lys were protected by Fmoc
(9-fluorenylmethyloxycarbonyl), and the hydroxy group of Tyr by 2-Br-Z (2-bromobenzyloxycarbonyl). Peptides were
assembled using Nα-Fmoc-protected amino acids and 1-hydroxybenzotriazole (HOBt)/1, 3-diisopropylcarbodiimide
(DIC)/N,N-Diisopropylethylamine
(DIEA)
or
1-hydroxybenzotriazole
(HOBt)/1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC•HCl)/4-dimethylaminopyridine (DMAP)
as coupling agents. The side chains of Dmt were also Fmoc-protected. The phenolic hydroxyl group of Dmt was
unprotected. Other side chain protection was as follows: tBu for Tyr and Cys, Boc for D-Lys, Trp, L-Phe and Orn, Pbf
for Arg and D-Arg. A 4-fold excess of building blocks (Fmoc-L-Pro-OH, Fmoc-Orn(Boc)-OH, Fmoc-D-Lys(Boc)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-L-Phe(Boc)-OH, Fmoc-Trp(Boc)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(tBu)-OH, Fmoc-Tyr-OH and Fmoc-Dmt-OH) and activating
agents was applied, and dry DMF was used as a solvent. Fmoc deprotections were carried out by treatment of the resin
with 95% piperidine in DMF, twice, for 30 min each. Fmoc-Dmt-OH was coupled with 1,3-diisopropylcarbodiimide
(DIC) and 1-hydroxybenzotriazole (HOBt) to avoid side reactions when uronium-based coupling reagents were used.
Final cleavage of the peptide, as well as the 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) side chain
protection group removal were accomplished by treatment with 95%(V/V) TFA/H2O cleavage conditions for 2 h.
Crude peptides were obtained after evaporation of the cleavage mixture, precipitated in cold Et2O, and purified by
reversed-phase high-performance liquid chromatography (HPLC). The purified peptides (> 95% purity) were then
lyophilized, and the molecular mass of peptides was analyzed by agreement with those obtained. Molecular weights
were subsequently confirmed by ESI–MS. Analytical data of the peptides are presented in Table 1.
Larval zebrafish (Danio rerio) were produced through paired mating of the AB wild-type strains adult fish from
the China Zebrafish Resource Center (Wuhan, China). Animals were tested at 5–7 days post-fertilization (dpf) and held
in an incubator in E3 embryo medium (EM, consisting of 5 mmol/L NaCl, 0.17 mmol/L KCl, 0.33 mmol/L CaCl2 and
0.33 mmol/L MgSO4; pH 7.2) at 28.5 oC, during treatments unless otherwise noted. Embryos/larvae were raised at 28.5
oC, at a density of 50 larvae per 100-mm2 petri dish. All experimental protocols were performed on animals in
accordance with the guidelines of the Animal Care Ethics Committee of Shenyang Pharmaceutical University Medical
Center and the National Institutes of Health.
To examine the potential toxicity of designed peptides, zebrafish larvae were incubated with each peptide at 100
or 50 μg/mL for 6 h (n = 12 for each group). After exposure, the larvae were observed under a stereomicroscope, and
lethal endpoints were recorded.
The protective effect of peptides against gentamicin was screened as we previously reported [12]. The evaluated
peptide (~75 μmol/L) was imposed on zebrafish for 1 h before acute exposure to gentamicin (200 μmol/L, 1 h),
followed by another 1 h recovery. For rapid assessment of hair cell survival, the larvae were immersed in 1 μmol/L of
DiOC2(3) for 30 min, then rinsed 4× with EM and anesthetized with 0.02% MS-222. DiOC2(3) labelling was then
evaluated on a Leica epifluorescent microscope (Chroma Technologies, Brattleboro, VT) for 4 neuromasts (SO2, SO3,
O1 and MI1). The area of labelled neuromasts was quantified per animal, summed to calculate one value per animal,
and averaged for each group (Image-Pro Plus). Results were presented as the mean area of neuromast, as a percentage
of the group treated only with EM. The treatment of gentamicin-only was used as negative control. To further
highlight the protective effects, we also used SYTOX Green (5 μmol/L, 1 min) for hair cell counting. The applied
protocol was conducted as descibed above.
To further compare the protective effects of optimal peptides, the larvae were pretreated with optimal peptides for
chronic exposure to gentamicin (6 h). Various concentrations of gentamicin (2, 5, 10, 15 μmol/L) were individually
administrated to larvae for chronic exposure after peptide pretreating for 1 h and cotreatment for 6 h. TMRE (1 nmol/L,
20 min) was applied to stain remaining neuromasts for dose-response function models and pharmacological evaluation.
Images were analyzed by Image-Pro Plus by drawing an area of interest (AOI) around individual neuromasts with
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