Design, Synthesis, and Biological Evaluation of Dexamethasone-Salvianolic Acid B Conjugates and Nanodrug Delivery against Cisplatin-Induced Hearing Loss

pubs.acs.org/jmc

Article

Design, Synthesis, and Biological Evaluation of Dexamethasone−

Salvianolic Acid B Conjugates and Nanodrug Delivery against

Cisplatin-Induced Hearing Loss

Ruiqin Ye, Lifang Sun, Jinghui Peng, Aixin Wu, Xiaozhu Chen, Lu Wen,* Chuan Bai,* and Gang Chen*

Cite This: J. Med. Chem. 2021, 64, 3115 −3130

Read Online

sı

*

Metrics & More

Article Recommendations

Supporting Information

ACCESS

ABSTRACT: Cisplatin (CDDP) is an extensively used chemotherapeutic agent but has a high incidence of severe ototoxicity.

Although a few molecules have entered clinical trials, none have been approved to prevent or treat CDDP-induced hearing loss by

the Food and Drug Administration. In this study, an amphiphilic drug−drug conjugate was synthesized by covalently linking

dexamethasone (DEX) and salvianolic acid B (SAL) through an ester or amide bond. The conjugates could self-assemble into

nanoparticles (NPs) with ultrahigh drug loading capacity and favorable stability. Compared with DEX, SAL, or their physical mixture

at the same concentrations, both conjugates and NPs showed enhanced otoprotection in vitro and in vivo. More importantly, the

conjugates and NPs almost completely restored hearing in a guinea pig model with good biocompatibility. Immunohistochemical

analyses suggested that conjugates and NPs activated the glucocorticoid receptor, which may work as one of the major mechanisms

for their protective eﬀects.

prospective, single-dose, safety phase I study of FX-322 for

severe sensorineural hearing loss in adults.⁹However, there are

no recommended regimens or FDA-approved drugs for the

prevention and treatment of CDDP-induced ototoxicity.¹⁰

Recent eﬀorts have provided renewed hope, with insights

into the potential targets and structure−activity relationship,

allowing development of therapeutics to prevent or treat

hearing loss. Amitriptyline and 7,8-dihydroxyﬂavone, small

molecule agonists of the TrkB receptor, were tested for the

protection of cochlear neural function caused by noise

exposure.¹¹Dabrafenib, a BRAF kinase inhibitor, repressed

ERK phosphorylation in cochlear cells and protects against

CDDP-induced HC death with oral delivery.¹²Glucocorticoids

INTRODUCTION

■

Drug-induced hearing loss mainly refers to the inner ear

damage caused by drugs that can damage the auditory nervous

system.^1,2Platinum anticancer drugs recorded a high incidence

of hearing loss in the clinic.³Cisplatin (CDDP)-induced

ototoxicity generally manifests in the form of irreversible,

progressive, and bilateral sensorineural hearing loss, which

causes the irreversible death of auditory cells by apoptosis and

necrosis and leads to the apoptosis of hair cells (HCs).⁴⁻⁶

Mammalian cochlear HCs are unable to regenerate once

damaged, thereby resulting in permanent hearing loss.⁷

Because these clinical manifestations seriously aﬀect the ability

of patients to communicate and their quality of life, protection

against CDDP-induced HC damage is an important issue.⁸

Eﬀorts have been directed at the development of new drugs for

hearing loss, and clinical trials have been conducted with some

therapies pending Food and Drug Administration (FDA)

approval. A multicenter phase II randomized controlled trial

studied intratympanic injection of N-acetylcysteine (NAC) for

prevention of CDDP-induced hearing loss. There is also a

Received: November 5, 2020

Published: March 5, 2021

https://dx.doi.org/10.1021/acs.jmedchem.0c01916

J. Med. Chem. 2021, 64, 3115−3130

3115

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 1. Synthetic route of DEX−SAL conjugate 1. Reagents and conditions: (a) butanedioic anhydride, DMF, DMAP, rt, 24 h; (b) N-Boc-

ethylenediamine, DMF, DCM, HOBt/DIC, rt, 24 h; (c) DCM, TFA, 0 °C, 1.5 h; (d) DMF, DCM, HOBt/EDCI, rt, 24 h.

(GCs) have been evaluated as potential otoprotective drugs,

based on their anti-inﬂammatory, ion homeostasis, and

immune suppression eﬀects.^13,14GCs are steroid hormones

secreted from the adrenal cortex in response to stress, which

act by binding to cytoplasmic glucocorticoid receptors

(GRs).^15,16GRs are strongly expressed in the stria vascularis,

HCs, and spiral ligament of the cochlea and cochlear nerve.¹⁷

Dexamethasone (DEX), a synthetic GC, can speciﬁcally bind

to the GR in the cochlea and thus stimulates the expression of

regulatory genes in the nucleus, accelerates the blood ﬂow of

the cochlea, and improves microcirculation.¹⁸DEX is used as a

therapeutic to counteract the inﬂammatory substrate in inner

ear disorders, including noise-induced deafness and drug-

induced hearing loss.^19,20However, frequent administration

and large doses are required to achieve the desired eﬃcacy

against CDDP-induced hearing loss, which may lead to severe

side eﬀects.^21,22The low aqueous solubility of DEX also

hindered its applications in clinical practice.²³

Salvianolic acid B (SAL) is the main active component in

the water-soluble extract from Danshen (Salvia miltiorrhiza),

and it possesses many bioactivities.²⁴SAL has been widely

used for the treatment of cardiovascular and cerebrovascular

diseases.²⁵The protective eﬀects of SAL appear to be mediated

via antiapoptotic, antioxidative, and anti-inﬂammatory func-

tions.²⁶⁻²⁹At the same time, our previous studies found that

SAL showed protective activity against drug-induced hearing

loss.³⁰SAL could eliminate excessive oxygen free radicals to

prevent lipid peroxidation reaction,³¹which exhibited

protective eﬀects against aminoglycoside antibiotics or

CDDP-induced ototoxicity by suppressing the production of

reactive oxygen species and the mitochondrial apoptotic

pathway.³²SAL is water-soluble and convenient to administer.

However, its eﬃcacy is limited by poor biomembrane

permeability, resulting in extremely low bioavailability.^33,34

Drug−drug conjugation entails linking two or multiple

bioactive molecules covalently. Each molecule becomes the

precursor or carrier of the other. It not only can retain the

bioactivity of each drug but also can achieve complementary or

synergistic functions.³⁵In a typical nanodrug delivery system,

the carrier is usually biologically inert and yet accounts for a

majority of the content in the system. Therefore, the drug

content is low.³⁶The self-assembly of the drug−drug conjugate

forms a high-drug content nanodelivery system without

additional carriers. This strategy could avoid the adverse

reactions caused by the use of carriers.^37,38The most

important thing is that the system is fully composed of drug

molecules, reaching nearly 100%, which provides a new way to

improve drug loading.³⁹

In this study, hydrophilic SAL and lipophilic DEX were

conjugated via an ester or amide bond to obtain amphiphilic

conjugate 1 or 2, respectively. Compared with SAL, the

conjugates had higher liposolubility, which facilitated the

3116

https://dx.doi.org/10.1021/acs.jmedchem.0c01916

J. Med. Chem. 2021, 64, 3115−3130

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 2. Synthetic route of DEX−SAL conjugate 2. Reagents and conditions: (a) ethanol, NaIO₄/H₂SO_4,rt, 24 h; (b) N-Boc-ethylenediamine,

DMF, DCM, HOBt/DIC, rt, 24 h; (c) DCM, TFA, 0 °C, 1.5 h; (d) DMF, DCM, HOBt/EDCI, rt, 24 h.

crossing of the biomembrane. The conjugates are expected to

activate GR, which was expressed in cochlear tissues, and to

achieve synergistic eﬀects. Therefore, it was anticipated to

prevent CDDP-induced hearing loss with low toxicity. Herein,

we report the synthesis and characterization of the amphiphilic

conjugates and their self-assembled NPs. The anti−ototoxicity

eﬃcacy and biocompatibility of the conjugates and NPs were

investigated. This carrier-free design oﬀers a promising strategy

for preventing CDDP-induced hearing loss.

yield). The other route was to oxidize DEX to give compound

2a (95.2% yield). Subsequently, the same experimental

procedures described above were employed to react with

SAL to obtain conjugate 2 (59.9% yield), which is shown in

Figure 2. The conjugates were puriﬁed by silica gel column

1

chromatography and characterized by H NMR, ¹³C NMR,

and HRMS (ESI). All of the signals corresponded to

characteristic groups in the compound, such as δ 207.0 for

−CO in conjugate 1, δ 174.0 for −CO− of −COOH in SAL, δ

156.1 and 131.1 for −CC in DEX, and δ 45.0 for −CH₂in

conjugates in the ¹³C NMR spectrum. The signal at δ 6.28 in

RESULTS AND DISCUSSION

■

1

the H NMR spectrum was J trans isomer > J cis isomer in

Design, Synthesis, and Characterization of DEX−SAL

Conjugates. Multifunctional conjugates that couple two

diﬀerent drug molecules must demonstrate tight binding to

targets, excellent biocompatibility, and good eﬃcacy. With

these goals in mind, DEX−SAL conjugates were designed.

DEX has three hydroxyl groups, among which the hydroxyl at

C₂₁was the most active and relatively easy to modify.⁴⁰

Therefore, the hydroxyl group at C₂₁was selected as the site of

conjugation with SAL. Of the two carboxyl groups in SAL, the

ﬂexible reversal oleﬁn bond was more active while the other

was less active due to the presence of the surrounding benzene

ring and phenolic hydroxyl group.⁴¹Hydrophobic DEX was

conjugated with hydrophilic SAL via diﬀerent linkages to study

the eﬀect of linkers on self-assembly and antiototoxic eﬃcacy.

The linker of the conjugates contained two amide bonds,

and the diﬀerence was that conjugate 1 also contained one

ester bond. Conjugate 1 was prepared according to the

procedures shown in Figure 1. DEX was ﬁrst reacted with

butanedioic anhydride to form compound 1a in a high yield of

90.3%. Compound 1a was combined with N-Boc-ethylenedi-

amine via an amide bond in the presence of N,N′-

diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole

(HOBt) to produce compound 1b in 90.1% yield. Then

compound 1b was deprotected in triﬂuoroacetic acid (TFA)

and dichloromethane (DCM) to generate key compound 1c in

87.2% yield. Finally, the amino group of compound 1c and the

carboxyl of SAL were connected to prepare conjugate 1 (65.9%

conjugates. In addition, the molecular formula obtained by

HRMS (ESI) was consistent with the theoretical formula.

Conjugate 1 was C₆₄H₆₇FN₂O₂₂, and conjugate 2 was

C₅₉H₆₁FN₂O₁₉. The purity (>98%) of conjugates was

determined by high-performance liquid chromatography

(HPLC) (for all data, see Figures S1 −S36).

Formation and Characterization of Conjugate NPs.

The inherent amphiphilicity of the synthesized DEX−SAL

conjugates could facilitate self-assembly into NPs in an

aqueous solution. A stable, white solution of ﬁnal conjugate

1 and a yellow solution of conjugate 2 were obtained at a

concentration of 2.5 mg/mL. The size and surface charge of

the self-assembled NPs were studied by dynamic light

scattering (DLS). A narrow unimodal distribution was

observed, and the average hydrodynamic diameters were

93.63 0.16 and 120.63 2.15 nm for conjugate 1 NPs and

conjugate 2 NPs, respectively (Figure 3A,B). The change in

size was mainly attributed to the stronger hydrophobic

interactions within conjugate 1, which resulted in a more

compact hydrophobic core.⁴²DLS also revealed the surface

charge of conjugate 1 NPs was −2.28

0.16 mV and the

1.43 mV

surface charge of conjugate 2 NPs was −21.46

(Figure 3C,D) in PBS (pH 7.4). A net charge carried by the

NPs was beneﬁcial for colloidal stability because mutual

repulsion can eﬀectively prevent aggregation.⁴³The morphol-

ogy of the aggregates was observed by transmission electron

3117

https://dx.doi.org/10.1021/acs.jmedchem.0c01916

J. Med. Chem. 2021, 64, 3115−3130

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 3. Characterization of DEX−SAL conjugate NPs. Particle size distribution of (A) conjugate 1 NPs and (B) conjugate 2 NPs by DLS. ζ

potentials of (C) conjugate 1 NPs and (D) conjugate 2 NPs determined by DLS. Morphology images of (E) conjugate 1 NPs and (F) conjugate 2

NPs by TEM. The scale bar was 100 nm.

microscopy (TEM), which conﬁrmed the formation of

spherical nanostructures. This implied both conjugates were

capable of self-assembling in aqueous solutions without any

exogenous excipients. The average size was approximately 60

nm (Figure 3E,F), which was smaller than that measured by

DLS due to the shrinkage of NPs in a dried state as a result of

TEM sample preparation.

The physiological stability is vital for biomedical application

of NPs. In the carrier-free NP system, the hydrophobic

interactions among conjugate molecules and electrostatic

repulsion among NPs increased the structural integrity of the

3118

https://dx.doi.org/10.1021/acs.jmedchem.0c01916

J. Med. Chem. 2021, 64, 3115−3130

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 4. In vitro stability and accumulative release of DEX−SAL conjugate NPs. Measurements of diameter and PDI during storage at 4 °C (A)

for 28 days of conjugate 1 NPs and (B) for 49 days of conjugate 2 NPs by DLS. Measurements of diameter and PDI in PBS containing 10% FBS

for 72 h of (C) conjugate 1 NPs and (D) conjugate 2 NPs. Measurements of diameter and PDI at diﬀerent pH values (5.0, 6.0, and 7.0) for 72 h of

(E) conjugate 1 NPs and (F) conjugate 2 NPs. (G) Release of conjugates from conjugate NPs in PBS at pH 7.4 and 37 °C. Data are expressed as

means the standard deviation (n = 3).

self-assembled NPs.⁴⁴To investigate the stability of the NPs in

PBS at 4 °C, the hydrodynamic diameter and polydispersity

index (PDI) were measured by DLS over time. As one can see

in Figure 4A, conjugate 1 NPs varied from 90.37 2.24 to

93.16 0.94 nm with a narrow PDI (0.14−0.15) for ≤21 days.

After that, the particle size gradually increased. In contrast,

conjugate 2 NPs did not show signiﬁcant changes in diameter

or PDI for 49 days (Figure 4B). This higher colloidal stability

was possibly due to higher net surface potentials.⁴⁵All results

demonstrated the long-term stability of both NPs at 4 °C. In

addition, the particle size and PDI of NPs in the PBS

containing 10% FBS exhibited no obvious change (Figure

3119

https://dx.doi.org/10.1021/acs.jmedchem.0c01916

J. Med. Chem. 2021, 64, 3115−3130

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 5. Anti−ototoxicity assay of the conjugates and NPs. (A) Eﬀect of CDDP-mediated decrease on ther viability of HEI-OC1 cells. The values

are expressed as means SD (***P < 0.001 vs CDDP; ^###P < 0.001 vs SAL, DEX, or the DEX/SAL mixture; n = 6). (B) Analysis of neuromast

HCs in zebraﬁsh after CDDP damage determined by staining with DASPEI. (C) Quantitative analysis of the mean ﬂuorescence area of zebraﬁsh

larvae using ImageJ. The data are presented as means SD (**P < 0.01; ***P < 0.001 vs CDDP; ^##P < 0.01; ^###P < 0.001 vs SAL, DEX, or the

DEX/SAL mixture; n = 7). (D) Mean ABR threshold against CDDP-induced hearing loss in a guinea pig model of the right ear. (E) Mean ABR

threshold of the left ear. (F) Images of HCs recorded with a confocal laser microscope, stained with FITC-phalloidin. The scale bar is 50 μm. (G)

Quantitative analysis of the numbers of OHCs at the basal turn determined by Zeiss software. The data are presented as means SD (***P <

0.001 vs CDDP; ^###P < 0.001 vs SAL, DEX, or the DEX/SAL mixture; n = 6).

3120

https://dx.doi.org/10.1021/acs.jmedchem.0c01916

J. Med. Chem. 2021, 64, 3115−3130

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

4C,D). These data demonstrated the good physical stability of

NPs. Furthermore, particle sizes and PDI were almost

unaltered when placed in solutions of diﬀerent pH values for

72 h in vitro (Figure 4E,F). Therefore, the NPs exhibited good

colloidal stability without any aggregation or precipitation at

various pH values in the absence of any excipients or

surfactants. Meanwhile, the in vitro release behaviors of

conjugates NPs were tested in PBS at pH 7.4, mimicking the

conditions in normal physiological tissues, and the release

proﬁles of conjugates from conjugate NPs are shown in Figure

4G. The release proﬁle showed an initial burst release during

the ﬁrst 6 h with approximately 50% of encapsulated conjugate

1 or 2 being released from conjugate NPs. Subsequently, the

rate of conjugate release decreased and reached 77% at 24 h.

At the end of the 4 day release study, the drugs released

plateaued at ∼80% for both NPs, which was consistent with

the release proﬁle of other typical NPs.⁴⁶

Anti−ototoxicity Assay. The protective eﬀects of the

conjugates and NPs against CDDP-induced ototoxicity were

examined using the House Ear Institute-Organ of Corti 1

(HEI-OC1) cell model. The 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) method and the

experimental samples were divided into 10 groups. As shown

in Figure 5A, the cell viabilities in the CDDP, SAL, DEX, and

DEX/SAL mixture groups were estimated to be 66.40

were measured to be 237.15 26.47, 268.04 49.09, 229.90

35.91, and 210.62

30.37 μm², respectively. These

ﬂuorescence areas increased by 60−100 μm², as compared to

those of DEX, SAL, and the DEX/SAL mixture, indicating that

the conjugates and NPs could signiﬁcantly prevent CDDP-

induced zebraﬁsh HC damage (P < 0.01; P < 0.001) (Figure

5C).

The hearing and vestibular systems of guinea pigs are very

similar to those of humans, and guinea pigs are frequently used

as animal models in the study of auditory systems and inner ear

diseases.^47,48To further test the eﬃcacy against CDDP-

induced hearing loss by the conjugates and NPs, guinea pigs

were randomly divided into 10 groups and DEX, SAL, the

DEX/SAL mixture, conjugates, and NPs (0.23 mg/kg) were

injected. All groups received two transtympanic administra-

tions, 1 h and 1 day prior to intraperitoneal injection of CDDP

(12 mg/kg). Conventional auditory brainstem response (ABR)

recording was used to monitor auditory function and

performed 3 days after CDDP administration. For the

CDDP, DEX, SAL, or DEX/SAL mixture group, the ABR

threshold was >90 dB in the entire frequency range while

pretreatment with conjugate 1 reduced the threshold to 52.02

3.59, 55.65 3.96, and 57.18 4.34 dB at frequencies of 4,

8, and 16 kHz, respectively. Pretreatment with conjugate 2,

conjugate 1 NPs, or conjugate 2 NPs showed threshold

reductions comparable to that of conjugate 1, illustrating that

all conjugates and NPs exhibited signiﬁcantly enhanced

protective eﬀects from CDDP-induced hearing loss and almost

entirely protected hearing ability (Figure 5D). Surprisingly,

from the experimental results, although the conjugates and

NPs were administered to the right ear, the hearing threshold

of the left ear resembled that of the right ear (Figure 5E),

indicating that conjugates and NPs could reach the

contralateral ear to protect hearing function. The cochlear

aqueduct may be the potential route of transfer to the

contralateral ear.^49,50

Furthermore, to investigate the morphological changes after

transtympanic administration of the conjugates and NPs in

these HCs, the basilar membrane of cochlea at the basal turn

was examined by ﬂuorescence microscopy. The cochlear HCs

were ﬂuorescently stained with FITC-phalloidin (green

ﬂuorescence), and the number of outer hair cells (OHCs) at

the basal turn was simultaneously quantiﬁed (Figure 5F).

Compared to the control group, the CDDP group exhibited

severe abnormalities and loss of HCs, indicating OHCs were

more susceptible to CDDP than inner hair cells (IHCs). As

shown in Figure 5G, the survival rates of HCs in the CDDP,

DEX, SAL, and DEX/SAL mixture groups were estimated to

be 33.78 2.75%, 41.38 4.01%, 44.76 3.88%, and 45.39

3.89%, respectively, whereas the rates in conjugate and NP

groups were all >93%, almost completely rescuing the HCs

from CDDP damage (P < 0.001). The results of HC survival

rates were consistent with those of the previous ABR

experiment. In comparison, this result of the positive group

was 91.52 2.60%, showing that DEX required a large dose to

provide a similar level of protection against CDDP-induced

hearing loss and was approximately 14.5-fold more than those

of guinea pig treated with the conjugates and NPs, respectively

(Figure 5G). Indeed, DEX was prescribed at large doses, which

could potentially cause harmful side eﬀects, including adrenal

insuﬃciency, gastrointestinal hemorrhage, and elevated blood

sugar levels.^21,22,51These results further demonstrated that the

eﬃcacy of the conjugates and NPs was the highest in all

1.32%, 62.78 2.08%, 63.56

1.82%, and 65.89

3.71%,

respectively. Upon pretreatment with conjugate 1 or conjugate

1 NPs for 1 h, followed by treatment with 40 μM CDDP for 24

h, the cell viabilities of conjugate 1 and conjugate 1 NP

pretreated groups were both approximately 72%. Furthermore,

the cell viabilities of conjugate 2 and conjugate 2 NP

pretreated groups were 76.31

1.90% and 74.18

2.13%,

respectively. In addition, the cell survival rate of the positive

group was markedly increased (P < 0.001), compared with that

of CDDP. These results indicated that pretreatment with the

conjugates or NPs signiﬁcantly restored the viability of HEI-

OC1 cells (P < 0.001), conﬁrming the protective eﬀect against

CDDP. In the meantime, no obvious diﬀerence was observed

between the conjugates and NPs. We suspected that the higher

level of intracellular accumulation of the conjugates and NPs

may have contributed to the better activity toward HEI-OC1

cells.

On the basis of the cytoprotective eﬀect, the in vivo

protective eﬀects of conjugates and NPs against CDDP-

induced ototoxicity on four neuromasts, namely, supraorbital

(SO1 and SO2), otic (O1), and occipital (OC1), in zebraﬁsh

were evaluated. This was done by examining the ﬂuorescence

area of 2-[4-(dimethylamino)styryl]-N-ethylpyridinium iodide

(DASPEI), which correlated well with cell viability. Treatment

with 700 μM CDDP led to cellular damage, evidenced by a

decreased DASPEI area. DEX, SAL, and the DEX/SAL

mixture did not protect against the CDDP-induced cellular

damage. In contrast, pretreatment of zebraﬁsh with conjugate

1, 2, or NPs protected against the cellular damage (Figure 5B).

Untreated control animals had an average DASPEI area of

375.33 25.38 μm², while the 700 μM CDDP only control

group had an average DASPEI area of 155.75 21.22 μm²in

the four neuromasts, showing signiﬁcant HC loss (P < 0.001).

In the SAL, DEX, and DEX/SAL mixture groups, the average

DASPEI areas were 164.78

153.89

30.37, 159.77

25.12, and

37.69 μm², respectively. While for pretreatment

zebraﬁsh larvae with conjugate 1, 2, or NPs, the ﬂuorescence

areas of conjugate 1, 2, conjugate 1 NPs, and conjugate 2 NPs

3121

https://dx.doi.org/10.1021/acs.jmedchem.0c01916

J. Med. Chem. 2021, 64, 3115−3130

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 6. Expression of GR in the cochlea. (A) Images of GR expression by immunohistochemistry. The scale bar is 100 μm. (B) Quantiﬁcation of

the tissue section. The data are presented as means SD (***P < 0.001 vs control; n = 6). (C) Schematic illustrating the proposed interactions of

the conjugates and NPs with GR. Abbreviations: SL&SV, spiral ligament and stria vascularis; SGCs, spiral ganglion cells; OC, organ of Corti.

groups, including the DEX, SAL, and DEX/SAL mixture

groups.

all observed sections, indicating a valid manner of quantiﬁca-

tion. Among the three cochlear tissue samples (Figure 6B), the

highest GR density was detected in SGCs, followed by SL&SV,

and the smallest quantity of GR was detected in OC. The

immunostaining results indicated that GRs were expressed in

the cochlea. Groups exposed to DEX had larger GR

immunostaining areas, which conﬁrmed that DEX could

reach the cochlea and increase the level of expression of

receptors (P < 0.001). Quantiﬁcation of GR expression in the

cochlear regions revealed a statistically signiﬁcant increase in

the GR staining area for the conjugate 1, 2, conjugate 1 NP,

and conjugate 2 NP groups compared with the control group

(P < 0.001). The results of this study suggested the conjugates

and NPs could bind to GR in cochlea and also promote

expression without compromising the binding aﬃnity or

receptor activation. Moreover, there was no observable

diﬀerence in the three cochlear tissues between the conjugate

and conjugate NP treatment groups, illustrating that the NPs

did not weaken its ability to activate the receptor.

Many pharmacological studies have shown that the classical

genomic pathway is major pathway for the action of GR.⁵⁹In

the genomic pathway, GCs diﬀuse through the cell membrane

and bind speciﬁc intracellular GRs to form a multiprotein

complex with heat shock proteins, immunophilins, and several

kinases. The activated GC/GR complex aﬀects gene tran-

scription and subsequent protein synthesis. GCs are known to

induce long-term changes in cellular function by this

mechanism.⁴⁸DEX can bind GR in the cytoplasm directly to

form a DEX/GR complex that will be actively transported from

GR Expression Studies. GRs have been identiﬁed in inner

ear tissues of several animal models.⁵²GCs, including DEX,

have been routinely prescribed for the treatment of various

inner ear pathologies. GC therapy is believed to modulate

cellular regulators of cochlear homeostasis after binding to the

cytoplasmic GR.⁵³The presence of such a receptor would

imply that GC could act directly on discrete inner ear tissue

regions. Direct activation of GR and the protective eﬀect of

DEX against noise-induced hearing loss had been demon-

strated in experimental animal models.^54,55In fact, it is known

that the number of GRs in a given tissue determines the

biologic responses of that tissue to GC.⁵⁶We sought to test

whether the conjugates and NPs interact with GR in guinea pig

inner ear tissues and inﬂuence the GR expression levels.

In this study, a rabbit anti-mouse GR antibody was used as

the ﬁrst antibody. For the negative control group, PBS buﬀer

was used instead of the primary antibody. To visualize GR

binding and study if GR expression was upregulated by the

conjugates and NPs, a dose of 1.67 mg/kg was used according

to the literature.^53,57The nucleus and cytoplasm were blue,

and the receptors appeared brownish yellow (Figure 6A).

According to the immunohistochemistry for the control group,

GRs were located in the organ of Corti (OC), spiral ganglion

cells (SGCs), and the spiral ligament and stria vascularis

(SL&SV) of the cochlea, which were consistent with the

results of ref 58. The immunostaining of the nucleus and

cytoplasm was stronger than that of the background tissue in

3122

https://dx.doi.org/10.1021/acs.jmedchem.0c01916

J. Med. Chem. 2021, 64, 3115−3130

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 7. In vivo biocompatibility evaluation. Analysis 120 hpf for zebraﬁsh embryos treated with conjugates and NPs of (A) the survival rate, (B)

the hatching rate, (C) the malformation rate, (D) embryonic development, (E) the body length, (F) and neuromast HCs by staining with DASPEI.

(G) Quantitative analysis of the mean ﬂuorescence area using ImageJ. The data are presented as means SD (n.s., P > 0.05 vs control; n = 20).

(H) Mean ABR threshold in a guinea pig model of the right ear. (I) Mean ABR threshold of the left ear. The data are presented as means SD

(n.s., P > 0.05 vs control; n = 6). (J) Histological examination of the cochlear tissues after transtympanic administration of conjugates and NPs.

Abbreviations: SL&SV, spiral ligament and stria vascularis; OC, organ of Corti; RM, Reissner’s membrane; TM, tectorial membrane; SGCs, spiral

ganglion cells.

3123

https://dx.doi.org/10.1021/acs.jmedchem.0c01916

J. Med. Chem. 2021, 64, 3115−3130

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

the cytoplasm to the nucleus, which induces a conformational

change in the GR without the use of a second messenger

system. The conformationally changed GR can bind to speciﬁc

DNA sequences and promote expression of certain proteins.

Indeed, an increased level of de novo synthesis of certain inner

ear proteins was detected after a given administration of DEX,

which suggested that DEX aﬀected the synthesis of metabolic

enzymes.⁶⁰In the guinea pig, DEX minimized hearing loss

signiﬁcantly when given prior to noise exposure, possibly by

preventing GR downregulation and thus stabilizing inner ear

function. The eﬀect of DEX on the auditory system may be

upregulation of GR through the rapid genomic transcription

pathway.⁶¹DEX had been reported to increase the level of

expression of GR in the inner ear in cases of noise-induced

hearing loss to protect the SGCs, SL&SV, and OC.⁶¹In the

meantime, DEX can also play an anti-inﬂammatory role and

regulate the immune system by interacting with GR, which

could also prevent hearing loss. The drug concentration in

perilymph within the cochlear samples was determined by

HPLC. The results showed that DEX was at a concentration of

97.31 6.02 μg/mL, conjugate 1 at a concentration of 133.56

2.96 μg/mL, and conjugate 2 at a concentration of 110.93

3.90 μg/mL, and animals treated with NPs exhibited

the SL&SV revealed clearly deﬁned nuclei and dense striae

with no cells bulging into the endolymphatic space in the

control group and pathological alternation in the inner ear

structure was not present in the experimental groups. The

structure and morphology were maintained well in the OC,

and no changes were found in the conjugate and NP groups

compared to the control group. Degeneration and myelin

sheath detachment were not observed in the control or

experimental groups in SGCs. On the basis of the results of our

observations, the conjugates and NPs did not cause obvious

histological changes in the inner ear, showing good

biocompatibility.

CONCLUSIONS

■

In summary, a hydrophobic drug was conjugated to a

hydrophilic drug via an ester or amide bond to form conjugates

1 and 2. The amphiphilic conjugates spontaneously self-

assembled into stable NPs in aqueous solution. The carrier-free

NPs exhibited a spherical morphology, a uniform particle size

distribution, a net negative surface charge, and a high drug

loading. Importantly, the eﬃcacy of conjugates and NPs on

CDDP-induced ototoxicity was demonstrated in vitro by using

the HEI-OC1 cell line and in vivo in zebraﬁsh and guinea pig

models. The conjugates and NPs almost completely restored

hearing at all tested frequencies, and the survival rates of HCs

were in accordance with the ABR results. From all of the anti−

ototoxicity results, no signiﬁcant diﬀerence was observed

between conjugates 1 and 2, suggesting that the diﬀerent

length of the linkage did not aﬀect the activity. Compared with

DEX, SAL, and their physical mixture, the conjugation of two

drugs exhibited synergetic and higher anti−ototoxicity activity.

It is noteworthy that an immunohistochemistry study revealed

conjugates and NPs could enhance the expression of GR,

which suggested that they may increase the pharmacological

eﬀects through targeting and activating the receptor. When

DEX was incorporated into the conjugates and NPs, its

binding to GR and associated bioactivity was not aﬀected.

Furthermore, histological assessment indicated that conjugates

and NPs had no adverse eﬀects and did not elicit inﬂammatory

responses in the cochlear tissue, preliminarily showing good

biosafety. In conclusion, the amphiphilic DEX−SAL con-

jugates and their NPs may be explored as a new way to prevent

drug-induced ototoxicity.

concentrations of 124.55

2.51 μg/mL (conjugate 1) and

101.57 3.38 μg/mL (conjugate 2) (Figure S37). The results

presented above indicated that the conjugates and NPs could

reach the cochlear tissue. Because the conjugates and NPs

activated the speciﬁc GR and increased its level of expression,

we speculated that the conjugates and NPs may exert

pharmacological eﬀects by inﬂuencing the speciﬁc DNA

sequences within the nucleus, and the interactions of

conjugates and NPs with GR and subsequent biological

processes are shown in Figure 6C. Speciﬁcally, we speculated

that DEX targeted the receptors to maintain cochlear function

while SAL exhibited antioxidant and antiapoptosis eﬀects to

prevent CDDP-induced hearing loss.

In Vivo Biocompatibility Evaluation. The biocompati-

bility of conjugates and NPs was assessed using zebraﬁsh

embryos and guinea pigs. Zebraﬁsh embryos were treated with

conjugates and NPs 120 h postfertilization (hpf). The

conjugate-treated group had a survival rate of ∼85%, while

NP-treated animals exhibited survival rates of 96.93% and

93.90% for conjugate 1 NPs and conjugate 2 NPs, respectively,

in comparison to a rate of 95% for the control group. There

was no signiﬁcant diﬀerence in survival rate (P > 0.05) (Figure

7A). Similarly, 120 hpf, no diﬀerences in the hatching,

malformation, body length, or mean ﬂuorescence area of

neuromasts were evident when compared with control

zebraﬁsh (P > 0.05) (Figure 7B,C,E−G). Zebraﬁsh embryos

exposed to conjugates or NPs showed normal development,

and no apparent phenotypic changes were observed (Figure

7D). Moreover, the biocompatibility of conjugate 1, conjugate

2, conjugate 1 NPs, or conjugate 2 NPs after transtympanic

injection was assessed by ABR, and histological features of

cochlear tissue on the OC, SL, RM, SV, TM, and SGCs were

further investigated. There were no signiﬁcant changes in the

ABR threshold over the entire frequency range (P > 0.05),

indicating that treatment of normal guinea pigs with conjugates

and NPs does not aﬀect hearing ability (Figure 7H,I). The

inﬂammatory response was not observed in the cochlear tissue

in any experimental group compared to the control group,

which veriﬁed that the conjugates and NPs were not toxic to

the cochlear tissue (Figure 7J). In addition, cross sections of

EXPERIMENTAL SECTION

■

General Experimental Procedure. DEX (8S, 9R, 10S, 11S, 13S,

14S, 16R, 17R) was purchased from Hubei Yuan Cheng Sai chuang

Technology Co., Ltd. (Hubei, China). SAL (7R, 8R, 8″R, 8″′R) was

obtained from Shanghai Li Ding Biotechnology Co., Ltd. (Shanghai,

China). The DEX/SAL mixture was ﬁxed in a 1:1 DEX:SAL molar

ratio. Succinic anhydride and sodium periodate were purchased from

Energy Chemical Co., Ltd. (Shanghai, China). 1-Ethyl-3-[3-

(dimethylamino)propyl]carbodiimide hydrochloride (EDCI), 1-hy-

droxybenzotriazole (HOBt), 4-(dimethylamino)pyrideine (DMAP),

N-Boc-ethylenediamine, and N,N′-diisopropylcarbodiimide (DIC)

were obtained from Shanghai Shaoyuan Co., Ltd. (Shanghai,

China). Sepha G-75 gel ﬁltration medium was purchased from

Shanghai Lanji Biotechnology Co., Ltd. (Shanghai, China). Flash

column chromatography was realized with 300−400 mesh silica gel

from Qingdao Haiyang Chemical Co., Ltd. (Qingdao, China).

Triﬂuoroacetic acid (TFA), ethyl acetate, methanol, dimethylforma-

mide (DMF), dichloromethane (DCM), and dimethyl sulfoxide

(DMSO) were obtained from Tianjin Zhiyuan Chemical Reagent Co.,

Ltd. (Tianjin, China). Bovine serum albumin (BSA) was purchased

3124

https://dx.doi.org/10.1021/acs.jmedchem.0c01916

J. Med. Chem. 2021, 64, 3115−3130

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

from Qi Yun Biotechnology Co., Ltd. (Guangzhou, China). N-Acetyl-

L-cysteine (NAC), DASPEI, and MTT were obtained from Sigma-

Aldrich (St. Louis, MO). FITC-phalloidin was supplied by the

Beyotime Institute of Biotechnology (Shanghai, China). The HEPES-

buﬀered Dulbecco’s modiﬁed eagle medium (DMEM) solution (pH

7.4, Gibco, 1×, sterile) was purchased from Life Technologies Co.,

Ltd. (Grand Island, NY). High-glucose DMEM and fetal bovine

serum (FBS) were supplied by Thermo Fisher Scientiﬁc (Waltham,

MA). Cisplatin (30 mg, 6 mL) was purchased from Jiangsu Haosen

Pharmaceutical Group Co., Ltd. (Jiangsu, China). The rabbit anti-

mouse GRs were obtained from Wuhan Servicebio Technology Co.,

Ltd. (Wuhan, China), and the horseradish peroxidase-labeled goat

anti-rabbit IgG was provided by Shanghai Rebiosci Biotech Co., Ltd.

(Shanghai, China). The reagents used in HPLC were chromatography

grade, and all other chemicals and reagents were analytical grade.

¹H NMR and ¹³C NMR spectra were recorded using a Varian

Mercury Plus 500 MHz spectrometer. Chemical shifts (δ) are

reported in parts per million for the solution of the compound in

CD₃OD, with the residual peak of the solvent as an internal reference.

J values are in hertz. The melting point (mp) was determined with the

XT-4 digital melting point apparatus (Beijing Tech Instrument Co.,

Ltd.). The mass spectra (MS) were recorded via ultra-high-

performance liquid chromatography-tandem mass spectrometry with

Thermo Fisher Scientiﬁc TSQ Quantum. High-resolution mass

spectra (HRMS) were recorded on an electrospray injection (ESI)

Thermo Fisher Scientiﬁc LTQ FTICR mass spectrometer. The purity

of all tested compounds was ≥98%, as estimated by HPLC analysis

performed on a LC-20A with a C18 column (4.6 mm × 150 mm) and

the following method: a mobile phase gradient from 45% MeOH/

buﬀer (0.1% formic acid/H₂O) to 50% MeOH/buﬀer (0.1% formic

acid/H₂O) for 6 min, 50% MeOH/buﬀer (0.1% formic acid/H₂O) to

58% MeOH/buﬀer (0.1% formic acid/H₂O) for 44 min, 58%

MeOH/buﬀer (0.1% formic acid/H₂O) to 45% MeOH/buﬀer (0.1%

formic acid/H₂O) for 2 min, 45% MeOH/buﬀer (0.1% formic acid/

H₂O) for 28 min, a ﬂow rate of 0.5 mL/min, and peak detection at

286 nm under ultraviolet radiation, injection volume of 20 μL. The

morphology of the conjugates NPs was studied using a model JEM-

3100F transmission electron microscope (JEOL). A small drop of the

sample solution (0.5 mg/mL) was sprayed onto the carbon-coated

copper grid, and then the grid immersed in liquid nitrogen and freeze-

dried in vacuum at −50 °C before measurement. The ζ potential and

DLS measurements were performed under a Malvern Zetasizer 3000

HS instrument (Malvern Instruments, Ltd.). All samples were

measured at a scattering angle of 90°. The DASPEI-stained area of

HCs was examined under a ﬂuorescence microscope (Leica Co.) and

measured using ImageJ (National Institutes of Health, Bethesda,

MD). The resulting slides were mounted and observed with a Zeiss

inverted ﬂuorescence microscope (Carl ZEISS AG) or a DMI 3000

camera (Leica Co.).

stirred at rt overnight. The puriﬁcation method was the same as that

of compound 1a to give compound 1b as a white solid (580 mg,

90.1% yield): H NMR (500 MHz, CD₃OD-d₄) δ 7.85 (s, 1H), 7.28

1

(d, J = 10.1 Hz, 1H), 6.73 (s, 1H), 6.21 (dd, J = 10.2, 1.9 Hz, 1H),

5.99 (s, 1H), 5.35 (dd, J = 4.9, 1.4 Hz, 1H), 5.11 (s, 1H), 5.02 (d, J =

17.6 Hz, 1H), 4.77 (d, J = 17.6 Hz, 1H), 4.21−4.08 (m, 1H), 3.04 (q,

J = 5.9 Hz, 2H), 2.95 (t, J = 6.2 Hz, 2H), 2.90−2.77 (m, 1H), 2.71−

2.54 (m, 3H), 2.43−2.24 (m, 4H), 2.07 (s, 4H), 1.83−1.71 (m, 1H),

1.69−1.51 (m, 2H), 1.47 (s, 3H), 1.36 (s, 8H), 1.05 (td, J = 8.1, 4.1

Hz, 1H), 0.87 (s, 3H), 0.77 (d, J = 7.2 Hz, 3H); ESI-MS m/z 657.36

[M + Na]⁺; HRMS(ESI) calcd for [M + Na]⁺C₃₃H₄₇FN₂NaO₉

657.3163, found 657.3170.

Compound 1c. Compound 1b (500 mg, 0.79 mmol) was dissolved

in DCM (8.0 mL) in a ﬂask, and TFA (1.2 mL) was added. The

reaction mixture was stirred at 0 °C for 1.5 h under an argon

atmosphere, and the solvent was evaporated under vacuum. After

column chromatography [5:1 (v/v) DCM/MeOH], compound 1c

1

was obtained as a white solid (367 mg, 87.2% yield): H NMR (500

MHz, CD₃OD-d₄) δ 7.40 (d, J = 10.1 Hz, 1H), 6.28 (dd, J = 10.1, 2.0

Hz, 1H), 6.07 (d, J = 1.6 Hz, 1H), 5.06−4.88 (m, 2H), 4.30−4.19 (m,

1H), 3.45 (td, J = 5.9, 3.6 Hz, 2H), 3.34 (s, 2H), 3.13 (m, 1H), 3.04

(t, J = 5.9 Hz, 3H), 2.81−2.65 (m, 4H), 2.57 (t, J = 7.0 Hz, 2H), 2.42

(m, 1H), 2.38 (m, 2H), 2.31 (d, J = 13.6 Hz, 1H), 2.26−2.11 (m,

1H), 1.91−1.82 (m, 1H), 1.73 (d, J = 12.7 Hz, 1H), 1.64 (dd, J =

13.8, 2.1 Hz, 1H), 1.58 (s, 3H), 1.56−1.42 (m, 2H), 1.28 (s, 1H),

1.19 (t, J = 4.1 Hz, 1H), 0.99 (d, J = 2.2 Hz, 3H), 0.85 (d, J = 7.3 Hz,

3H); ESI-MS m/z 535.41 [M + H]⁺; HRMS(ESI) calcd for [M + H]⁺

C₂₈H₄₀FN₂O₇535.2820, found 535.2827.

Conjugate 1. Compound 1c (375 mg, 0.70 mmol), SAL (414 mg,

0.58 mmol), HOBt (120 mg, 0.88 mmol), and EDCI (169 mg, 0.88

mmol) were all placed in a ﬂask and dissolved in DMF (6.0 mL),

stirring at rt for 24 h. The solvent was removed in vacuo, and after

column chromatography [5:1 (v/v) DCM/MeOH], conjugate 1

(mixture of cis and trans isomers) was obtained as a yellow solid (571

mg, 65.9% yield, 98.2% pure): isolated pure cis isomer, 50 mg; isolated

pure trans isomer, 55 mg.

cis Isomer (1). mp 234.5−235.1 °C; ¹H NMR (500 MHz, CD₃OD-

d₄) δ 7.87 (d, J = 8.8 Hz, 1H), 7.74 (d, J = 7.9 Hz, 1H), 7.54 (d, J =

15.1 Hz, 2H), 7.40 (d, J = 10.1 Hz, 1H), 7.18 (d, J = 8.5 Hz, 1H),

6.89−6.50 (m, 9H), 6.36 (d, J = 8.0 Hz, 1H), 6.28 (d, J = 5.5 Hz,

1H), 6.07 (s, 1H), 5.74 (d, J = 4.6 Hz, 1H), 5.19 (s, 2H), 4.97 (d, J =

17.7 Hz, 2H), 4.52 (d, J = 4.6 Hz, 1H), 4.24 (d, J = 4.6 Hz, 1H), 3.21

(s, 3H), 3.07 (s, 1H), 2.98 (d, J = 11.2 Hz, 3H), 2.86 (s, 1H), 2.72−

2.65 (m, 2H), 2.48 (s, 1H), 2.40 (s, 1H), 2.29 (d, J = 13.7 Hz, 1H),

2.20 (d, J = 8.4 Hz, 1H), 1.99−1.79 (m, 2H), 1.67 (dd, J = 34.7, 12.7

Hz, 3H), 1.60−1.51 (m, 4H), 1.52 (dd, J = 13.0, 7.7 Hz, 2H), 1.30 (d,

J = 7.7 Hz, 2H), 1.17 (s, 2H), 0.98 (s, 3H), 0.83 (d, J = 7.2 Hz, 3H);

¹³C NMR (126 MHz, CD₃OD-d₄) δ 207.0, 207.0, 174.0, 173.9, 173.8,

172.8, 172.7, 171.2, 171.1, 156.1, 156.1, 149.2, 146.6, 146.5, 146.0,

145.9, 145.2, 145.1, 144.7, 144.1, 134.0, 133.9, 131.1, 129.8, 129.0,

128.6, 126.5, 126.3, 125.1, 124.7, 123.1, 122.3, 121.7, 118.4, 118.1,

117.8, 117.2, 116.4, 113.5, 103.1, 101.7, 92.4, 88.3, 76.1, 76.0, 73.1,

72.8, 69.8, 58.5, 45.0, 37.2, 35.6, 35.5, 33.4, 32.2, 31.5, 31.5, 30.2,

30.1, 28.8, 23.7, 23.7, 17.1, 15.2; ESI-MS m/z 1233.41 [M − H]⁻;

HRMS(ESI) calcd for [M − H]⁻C₆₄H₆₆FN₂O₂₂1233.4170, found

1233.4107.

Syntheses of 1a−c. 1 (1a). DEX (549 mg, 1.40 mmol) was

dissolved in DMF (1.0 mL), and butanedioic anhydride (250 mg, 2.50

mmol) and DMAP (330 mg, 2.70 mmol) were dissolved in DCM (6.0

mL); then the mixture was stirred at rt for 24 h under an argon

atmosphere. The solvent was evaporated in vacuum, and then the

mixture was extracted with ethyl acetate and washed three times with

brine (30.0 mL). The solvent was removed under vacuum, and the

residue was puriﬁed using silica gel column chromatography [10:1 (v/

v) DCM/MeOH] to obtain compound 1a as a white solid (622 mg,

trans Isomer (1). mp 234.8−235.3 °C; ¹H NMR (500 MHz,

CD₃OD-d₄) δ 7.87 (d, J = 8.3 Hz, 1H), 7.74 (d, J = 8.3 Hz, 1H), 7.43

(m, 2H), 7.41 (d, J = 10.1 Hz, 1H), 7.38−7.31 (m, 1H), 7.14 (d, J =

8.4 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.77 (s, 1H), 6.74 (d, J = 8.0

Hz, 1H), 6.70 (dd, J = 4.5, 3.2 Hz, 2H), 6.66 (d, J = 7.3 Hz, 1H), 6.55

(t, J = 8.9 Hz, 2H), 6.31 (d, J = 7.8 Hz, 1H), 6.28 (d, J = 10.1 Hz,

1H), 6.07 (s, 1H), 5.86 (s, 1H), 5.19 (m, 2H), 4.95 (d, J = 17.7 Hz,

2H), 4.39 (d, J = 3.6 Hz, 1H), 4.24 (d, J = 10.1 Hz, 1H), 4.10 (q, J =

7.1 Hz, 1H), 3.24 (s, 2H), 2.99 (d, J = 6.5 Hz, 2H), 2.66 (dd, J = 17.0,

12.6 Hz, 3H), 2.50−2.41 (m, 2H), 2.33 (d, J = 12.4 Hz, 1H), 2.32−

2.25 (m, 1H), 2.23−2.13 (m, 2H), 2.01 (s, 1H), 1.86 (dd, J = 12.7,

6.0 Hz, 2H), 1.74−1.57 (m, 3H), 1.56 (s, 2H), 1.53−1.44 (m, 2H),

1.30 (dd, J = 10.3, 5.9 Hz, 2H), 1.24 (t, J = 7.1 Hz, 1H), 1.15 (ddd, J

= 34.8, 17.3, 13.6 Hz, 2H), 0.97 (d, J = 7.5 Hz, 3H), 0.83 (d, J = 7.3

1

90.3% yield): H NMR (500 MHz, CD₃OD-d₄) δ 7.40 (s, 1H), 6.29

(s, 1H), 6.08 (s, 1H), 5.03−4.92 (m, 2H), 4.26 (dd, J = 10.6, 1.6 Hz,

1H), 3.02 (s, 1H), 2.72 (s, 3H), 2.62 (d, J = 1.9 Hz, 2H), 2.40 (s,

2H), 2.30 (s, 1H), 2.22 (s, 1H), 2.15 (s, 3H), 1.87 (s, 1H), 1.71 (m,

2H), 1.59 (s, 3H), 1.53 (dd, J = 12.9, 5.0 Hz, 1H), 1.19 (s, 1H), 1.01

(s, 3H), 0.86 (d, J = 7.3 Hz); ESI-MS m/z 515.35 [M + Na]⁺;

HRMS(ESI) calcd for [M + H]⁺C₂₆H₃₄FO₈493.2238, found

493.2253.

Compound 1b. Compound 1a (500 mg, 1.02 mmol) and HOBt

(206 mg, 1.52 mmol) were dissolved in DMF (1.2 mL), and N-Boc-

ethylenediamine (195 mg, 1.22 mmol) and DIC (192 mg, 1.52

mmol) in DCM (5.0 mL) were added. The reaction mixture was

3125

https://dx.doi.org/10.1021/acs.jmedchem.0c01916

J. Med. Chem. 2021, 64, 3115−3130

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Hz, 3H); ¹³C NMR (126 MHz, CD₃OD-d₄) δ 207.0, 206.9, 174.1,

174.0, 172.3, 172.3, 171.2, 171.0, 168.5, 156.1, 156.1, 148.9, 146.8,

146.8, 146.6, 146.1, 145.9, 145.2, 145.0, 144.9, 142.9, 133.4, 133.4,

131.0, 130.7, 129.8, 128.6, 128.4, 126.7, 126.3, 125.1, 124.6, 124.6,

121.9, 118.6, 118.4, 117.5, 117.3, 116.4, 113.5, 103.1, 101.7, 92.4,

88.5, 77.0, 75.8, 73.1, 72.8, 69.8, 57.4, 45.0, 37.2, 35.6, 35.5, 33.3,

32.2, 31.6, 30.7, 30.2, 28.8, 23.7, 23.6, 17.1, 15.2; ESI-MS m/z

1233.41 [M − H]⁻; HRMS(ESI) calcd for [M − H]⁻C₆₄H₆₆FN₂O₂₂

1233.4170, found 1233.4105.

6.7, 4.8 Hz, 1H), 6.28 (d, J = 4.8 Hz, 1H), 6.07 (d, J = 5.7 Hz, 1H),

5.56 (d, J = 4.4 Hz, 1H), 5.27 (s, 1H), 5.11 (s, 1H), 4.60 (d, J = 4.3

Hz, 1H), 4.24 (d, J = 11.7 Hz, 1H), 4.14 (d, J = 13.8 Hz, 1H), 3.26−

3.18 (m, 1H), 3.06 (d, J = 11.6 Hz, 2H), 2.98 (dd, J = 21.8, 8.0 Hz,

1H), 2.94−2.90 (m, 1H), 2.86 (s, 1H), 2.76−2.65 (m, 1H), 2.38 (dd,

J = 15.0, 10.8 Hz, 1H), 2.17−2.07 (m, 1H), 1.91−1.81 (m, 1H),

1.80−1.63 (m, 1H), 1.63−1.55 (m, 4H), 1.53−1.45 (m, 1H), 1.41

(dd, J = 15.6, 8.7 Hz, 2H), 1.27 (d, J = 21.4 Hz, 2H), 1.22 (d, J = 5.6

Hz, 1H), 1.08 (t, J = 14.3 Hz, 2H), 0.98 (d, J = 16.8 Hz, 3H), 0.89

(dd, J = 7.3, 2.7 Hz, 1H), 0.83 (d, J = 7.0 Hz, 3H); ¹³C NMR (126

MHz, CD₃OD-d₄) δ 189.5, 189.4, 176.5, 172.9, 171.8, 171.7, 171.6,

156.7, 149.6, 147.0, 146.8, 146.4, 146.2, 145.6, 145.4, 145.1, 144.4,

134.2, 131.4, 130.8, 130.3, 130.1, 129.4, 128.9, 126.8, 126.6, 125.0,

123.4, 122.6, 122.3, 118.7, 118.4, 118.1, 118.1, 116.8, 116.4, 113.9,

103.6, 102.2, 88.7, 73.6, 73.3, 58.9, 45.1, 41.1, 40.2, 38.9, 37.1, 36.6,

36.1, 36.0, 33.7, 32.6, 32.4, 29.1, 24.0, 23.9, 18.2, 15.6; ESI-MS m/z

1119.38 [M − H]⁻; HRMS(ESI) calcd for [M − H]⁻C₅₉H₆₀FN₂O₁₉

1119.3774, found 1119.3787.

Syntheses of 2a−c. 2 (2a). DEX (392 mg, 1.00 mmol) was

dissolved in ethanol, and sodium periodate (257 mg, 1.20 mmol) in a

sulfuric acid solution (0.12 mol/L, 31.0 mL) was added, stirring at rt

for 24 h. The solvent was evaporated in vacuum, and the residue was

adjusted with sodium hydroxide (1.0 mol/L) to pH 12 by washing

with DCM. The solution was adjusted with hydrochloric acid (1.0

mol/L) to pH 3 and extracted with ethyl acetate. The solvent was

removed under vacuum to obtain compound 2a as a white solid (360

1

mg, 95.2% yield): H NMR (500 MHz, CD₃OD-d₄) δ 7.41 (d, J =

trans Isomer (2). mp 235.8−236.7 °C; ¹H NMR (500 MHz,

CD₃OD-d₄) δ 7.52−7.38 (m, 3H), 7.35 (d, J = 7.6 Hz, 1H), 7.09 (d, J

= 8.4 Hz, 1H), 6.74 (ddd, J = 23.8, 23.0, 8.2 Hz, 8H), 6.57 (d, J = 10.5

Hz, 1H), 6.55 (dd, J = 33.0, 7.1 Hz, 1H), 6.28 (d, J = 10.3 Hz, 1H),

6.15 (d, J = 15.7 Hz, 1H), 6.08 (s, 1H), 5.91 (s, 1H), 5.20 (s, 2H),

4.39 (s, 1H), 4.23 (t, J = 12.6 Hz, 1H), 4.10 (q, J = 7.1 Hz, 1H),

3.28−3.23 (m, 2H), 3.05−2.91 (m, 1H), 2.87 (s, 1H), 2.76−2.75 (m,

1H), 2.76−2.65 (m, 1H), 2.50−2.31 (m, 1H), 2.26−2.07 (m, 1H),

2.01 (s, 1H), 1.91−1.80 (m, 1H), 1.79−1.72 (m, 1H), 1.66 (d, J =

11.7 Hz, 1H), 1.56 (s, 3H), 1.51−1.39 (m, 3H), 1.30 (s, 1H), 1.25 (t,

J = 7.0 Hz, 2H), 1.11 (d, J = 21.0 Hz, 2H), 1.04 (s, 3H), 0.91 (d, J =

7.2 Hz, 1H), 0.84 (d, J = 7.1 Hz, 3H); ¹³C NMR (126 MHz, CD₃OD-

d₄) δ 189.2, 189.1, 172.6, 171.5, 171.3, 168.6, 168.5, 156.4, 149.0,

146.9, 146.6, 146.2, 146.0, 145.3, 144.9 (overlap, 2C), 143.1, 136.4,

133.8, 133.7, 131.0, 130.5, 129.7, 128.6, 126.7, 126.3, 125.0, 124.7,

121.8, 121.6, 118.8, 118.4, 117.6, 117.4, 116.5, 116.3, 113.4, 103.3,

101.9, 88.5, 73.2, 72.9, 57.0, 44.8, 41.4, 39.8, 38.4, 36.9, 36.2, 35.8,

35.7, 33.3, 32.3, 32.2, 28.8, 23.6, 23.6, 17.9, 15.3; ESI-MS m/z

1119.38 [M − H]⁻; HRMS(ESI) calcd for [M − H]⁻C₅₉H₆₀FN₂O₁₉

1119.3774, found 1119.3789.

Preparation and Characterization of NPs. Conjugate 1 (10

mg) was dissolved in ethanol (1.0 mL) and added to PBS (4.0 mL,

pH 7.4), stirring at rt for 90 min. Subsequently, the ethanol was

removed under vacuum to give conjugate 1 NPs. The processing

method of conjugate 2 NPs was similar to that for conjugate 1 NPs,

which was stirred for 120 min. The particle size, PDI, and ζ potential

of conjugate NPs were determined with a Zetasizer Nano-ZS

instrument, and triplicate measurements for each sample were

taken. Conjugate NPs were negatively stained with a sodium

phosphotungstate solution and scanned with a transmission electron

microscope to examine the morphology.

Stability of NPs. The stability of conjugate NPs (2.5 mg/mL) was

studied by monitoring the particle size and PDI. The solution of the

conjugate 1 NPs was stored at 4 °C in a refrigerator for 28 days, and

that of conjugate 2 NPs for 49 days. At diﬀerent day intervals (0, 0.5,

1, 7, 14, 21, 28, 35, and 49 days), the average size and PDI were

determined. At the same time, the conjugate NPs were incubated in

PBS (pH 7.4) containing 10% FBS at 37 °C for 72 h. In addition, the

stability of the solution of conjugate NPs with diﬀerent pH values

(5.0, 6.0, and 7.0) at 37 °C was investigated and the particle diameter

and PDI were measured after time intervals of 0, 12, 24, 48, and 72 h.

In Vitro Drug Release Studies. The release proﬁles of

conjugates from NPs were studied using PBS as the release medium.

The solution of conjugate NPs (2.5 mg/mL, 1.0 mL) was placed in a

dialysis bag (molecular weight cutoﬀ of 2000 Da) and in PBS (90.0

mL, pH 7.4) while being continuously shaken at 100 rpm and

incubated at 37 °C, respectively, measuring at predetermined time

intervals (0.5, 1, 2, 4, 8, 12, 24, 36, 48, 72, and 96 h). At set time

points, the external buﬀer (0.5 mL) solution was withdrawn and

replaced with fresh PBS (0.5 mL) and samples were analyzed by

HPLC for the concentration of released conjugate 1 or 2.

11.0 Hz, 1H), 6.28 (dt, J = 10.1, 1.8 Hz, 1H), 6.07 (d, J = 1.8 Hz,

1H), 4.27 (d, J = 10.8 Hz, 1H), 3.02 (d, J = 1.5 Hz, 1H), 2.74 (d, J =

6.2 Hz, 1H), 2.49 (m, 1H), 2.43 (d, J = 5.5 Hz, 2H), 2.23−2.11 (m,

2H), 1.97−1.85 (m, 1H), 1.84−1.70 (m, 1H), 1.60 (s, 1H), 1.58 (m,

4H), 1.54 (dd, J = 12.8, 5.0 Hz, 1H), 1.50 (m, 1H), 1.31−1.19 (m,

1H), 1.14 (d, J = 1.5 Hz, 3H), 0.94 (dd, J = 7.3, 1.6 Hz, 3H); ESI-MS

m/z 401.25 [M + Na]⁺; HRMS(ESI) calcd for [M + H]⁺C₂₁H₂₈FO₅

379.1921, found 379.1927.

Compound 2b. Compound 2a (600 mg, 1.59 mmol) and HOBt

(322 mg, 2.38 mmol) were dissolved in DCM (5.0 mL), and N-Boc-

ethylenediamine (305 mg, 1.90 mmol) and EDCI (301 mg, 1.57

mmol) in DMF (1.0 mL) were added, stirring at rt overnight. The

extraction method was the same as that for compound 1a. The solvent

was removed under vacuum, and the residue was puriﬁed using silica

gel column chromatography [7:1 (v/v) DCM/MeOH] to obtain

compound 2b as a white solid (728 mg, 88.2% yield): ¹H NMR (500

MHz, CD₃OD-d₄) δ 7.87 (dd, J = 8.6, 3.8 Hz, 1H), 7.73 (d, J = 8.3

Hz, 1H), 7.57 (q, J = 5.8 Hz, 1H), 7.50 (td, J = 8.0, 4.1 Hz, 1H), 7.41

(d, J = 10.2 Hz, 1H), 6.27 (d, J = 10.1 Hz, 1H), 6.07 (s, 1H), 4.24 (d,

J = 11.9 Hz, 1H), 3.34 (d, J = 5.2 Hz, 1H), 3.24−3.07 (m, 3H), 2.98

(d, J = 4.9 Hz, 2H), 2.85 (d, J = 4.7 Hz, 2H), 2.73 (m, 1H), 2.54−

2.35 (m, 2H), 2.18 (dd, J = 20.3, 12.3 Hz, 2H), 1.87 (m, 1H), 1.76

(dd, J = 11.6, 3.6 Hz, 1H), 1.60 (d, J = 4.5 Hz, 3H), 1.45 (s, 8H), 1.21

(s, 1H), 1.09 (s, 3H), 0.91 (d, J = 7.3 Hz, 3H); ESI-MS m/z 543.08

[M + Na]⁺; HRMS(ESI) calcd for [M + Na]⁺C₂₈H₄₁FN₂NaO₆

543.2846, found 543.2862.

Compound 2c. Compound 2b (450 mg, 0.97 mmol) was dissolved

in DCM (4.0 mL), and TFA (1.0 mL) was added, stirring at 0 °C for

1.5 h. The processing method was the same as that for compound 1c

1

to give compound 2c as a white solid (328 mg, 90.2% yield): H

NMR (500 MHz, CD₃OD-d₄) δ 7.41 (d, J = 10.2 Hz, 1H), 6.28 (d, J

= 10.2 Hz, 1H), 6.07 (s, 1H), 5.48 (d, J = 4.6 Hz, 1H), 4.24 (d, J =

10.1 Hz, 1H), 3.50 (m, 1H), 3.34 (d, J = 5.2 Hz, 1H), 3.13 (s, 1H),

3.01−2.88 (m, 2H), 2.72 (m, 1H), 2.48 (m, 1H), 2.38 (d, J = 13.5 Hz,

2H), 2.23 (s, 1H), 2.21−2.12 (m, 2H), 1.88 (m, 1H), 1.76 (dd, J =

23.5, 12.0 Hz, 1H), 1.58 (d, J = 4.4 Hz, 3H), 1.53 (d, J = 4.3 Hz, 1H),

1.51 (d, J = 4.5 Hz, 1H), 1.46 (d, J = 14.2 Hz, 1H), 1.28 (s, 1H), 1.21

(m, 1H), 1.07 (d, J = 4.0 Hz, 3H), 0.89 (t, J = 5.8 Hz, 3H); ESI-MS

m/z 421.37 [M + H]⁺; HRMS(ESI) calcd for [M + H]⁺

C₂₃H₃₄FN₂O₄421.2503, found 421.2512.

Conjugate 2. Compound 2c (266 mg, 0.63 mmol), SAL (420 mg,

0.58 mmol), HOBt (120 mg, 0.88 mmol), and EDCI (169 mg, 0.88

mmol) were placed in a ﬂask and dissolved in DMF (6.0 mL), stirring

at rt for 24 h. The solvent was removed in vacuo, and after column

chromatography [5:1 (v/v) DCM/MeOH], conjugate 2 (mixture of

cis and trans isomers) was obtained as a yellow solid (425 mg, 59.9%

yield, 99.1% pure): isolated pure cis isomer, 42 mg; isolated pure trans

isomer, 45.5 mg.

cis Isomer (2). mp 235.9−236.8 °C; ¹H NMR (500 MHz, CD₃OD-

d₄) δ 7.56−7.40 (m, 2H), 7.35 (dd, J = 14.6, 9.0 Hz, 2H), 7.16 (d, J =

8.5 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 6.75−6.69 (m, 3H), 6.68−6.64

(m, 2H), 6.61−6.56 (m, 3H), 6.44 (d, J = 7.5 Hz, 1H), 6.30 (dd, J =

3126

https://dx.doi.org/10.1021/acs.jmedchem.0c01916

J. Med. Chem. 2021, 64, 3115−3130

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Cell Culture and Viability Assay. The HEI-OC1 cell line is a

widely used auditory HC line derived from the cochlea of the

immortomouse.⁶²Cells were maintained in high-glucose DMEM,

supplemented with 10% FBS at 33 °C, in a humidiﬁed incubator with

10% CO₂. Cell viability was measured using the MTT assay as

described previously.⁶³HEI-OC1 cells were seeded at a density of 1 ×

10⁴cells/well in 96-well plates and cultured overnight. The conjugates

were dissolved in DMSO. Dilutions from conjugates and NP solutions

were then performed in cell culture medium for the cellular assays. To

investigate the eﬀect of conjugates and conjugate NPs on cell viability,

HEI-OC1 cells were pretreated with DEX (90 μM), SAL (90 μM), a

DEX/SAL mixture (90 μM), conjugate 1 (80 μM), 2 (90 μM),

conjugate 1 NPs (80 μM), or 2 NPs (90 μM) for 1 h and then co-

treated with CDDP (40 μM) for 24 h. NAC (1 mM) was used as a

positive control. Then, an MTT solution (5.0 mg/mL) was added to

each cell culture, followed by incubation for 4 h at 33 °C with 10%

CO₂. Lastly, the MTT assay reached completion after the addition of

DMSO (100 μL/well) and mixed for 10 min. The absorbance of each

well was determined by a microplate spectrophotometer at 490 nm,

and assays were performed in triplicate. The relative amount of

inhibition of cell growth was calculated as follows:

transtympanically administered twice at a dose of 0.23 mg/kg, 24 and

1 h before CDDP injection, while the positive group was treated with

DEX (3.33 mg/kg).

ABR Experiment. Auditory function was measured 72 h after

administration of CDDP by ABR recording. The guinea pigs were

anesthetized with pentobarbital sodium (30 mg/kg) by intra-

peritoneal injection. Three needle electrodes were inserted sub-

dermally at the vertex (active), under the left ear (reference), and in

the back (ground). Free-ﬁeld pure tone stimuli were generated, and

ABR recordings were digitized using custom software. Tone-burst

stimuli with a 1 ms rise or fall time were presented at 4, 8, and 16 kHz

at a rate of 16.0 s⁻¹. The hearing threshold was deﬁned as the lowest

stimulation decibel level to evoke a positive wave in the response

trace.⁶⁵

Assessment of HCs. Cochleae of guinea pigs were rapidly

harvested after they had been anesthetized by intraperitoneal

injection, perfused with 4% paraformaldehyde in PBS, and further

immersed in the ﬁxative overnight at 4 °C. The cochlear basement

membrane was incubated in 0.1% Triton X-100 for 30 min and

washed three times with PBS. Specimens were counterstained with

2% BSA and 0.1% Triton X-100 and then diluted with FITC-

phalloidin at a ratio of 1:100 for 50 min. The tissues were mounted on

glass slides with antiﬂuorescent quencher dripping. Cochlear samples

were observed and imaged with a confocal laser microscope. HCs

were counted in six randomly selected 200 μm lengths of the basal

turn. The percentage of viable hair cells was calculated as the number

of viable HCs divided by the total number of HCs (viable HCs and

missing HCs).

Immunohistochemical Analysis. The guinea pigs were assigned

to seven groups (n = 6). The negative and control groups were

subjected to transtympanic injection of saline, while the other ﬁve

groups were DEX, conjugate 1, conjugate 2, conjugate 1 NPs, and

conjugate 2 NPs. Brieﬂy, a DEX solution, conjugates, and NPs with

equivalent DEX (1.67 mg/kg) received transtympanic administration

for 30 min. Animals were decapitated under deep anesthesia, and the

bubbles were quickly removed for ﬁxation. The cochleae were

decalciﬁed and prepared in paraﬃn sections. Sections with a 4 μm

thickness were deparaﬃnized with toluene and rehydrated by serially

graded ethanol solutions. After being washed three times in PBS, the

sections were autoclaved in 10 mM citrate buﬀer for 10 min at 121

°C. After endogenous peroxidase activity had been blocked with 0.3%

H₂O₂in methanol for 25 min at rt, the sections were preincubated

with 3% BSA in PBS for 30 min and the rabbit anti-mouse GRs were

overlaid overnight at 4 °C. Slides were consecutively incubated with

horseradish peroxidase-labeled goat anti-rabbit IgG (500 μg/mL) and

1% BSA in PBS for 1 h to block nonspeciﬁc reaction with the ﬁrst

antibody and ﬁnally DAB for 5 min. The negative control group was

performed for each specimen using the same method but without the

use of the primary antibody. The slides were counterstained with

hematoxylin, dehydrated in a graded alcohol series, cleared in xylene,

and examined with a microscope to observe the expression of GR. In

addition, after transtympanic injection of 1.67 mg of DEX, conjugates,

or NPs per kilogram, the drug content in perilymph was determined

to be 0.5, 1, 4, 8, and 24 h; the perilymph sampling process was

described in detail in previous studies.⁶⁶

cell viability (%) = (A_experiment− A_blank)/(A_control− A_blank

× 100%

)

Zebraﬁsh Husbandry and Drug Treatments. Adult zebraﬁsh

was obtained from the School of Pharmaceutical Sciences, Sun Yat-

sen University. Zebraﬁsh (Danio rerio) embryos were produced by

paired mating of AB wild-type adult ﬁsh in zebraﬁsh facilities at the

School of Pharmacy, Guangdong Pharmaceutical University. All of the

animal experiments were conducted according to the Guidelines for

the Care and Use of Laboratory Animals and received ethical approval

from the Institutional Animal Ethical Care Committee of Guangdong

Pharmaceutical University. Experiments were performed 5−6 days

postfertilization (dpf), and larval zebraﬁsh maintained at 28 °C in a

deﬁned embryo medium (EM) containing MgSO₄(1 mM), KH₂PO₄

(120 μM), Na₂HPO₄(74 μM), CaCl₂(1 mM), KCl (500 μM), NaCl

(15 mM), and NaHCO₃(500 μM) in distilled water at pH 7.2.

Neuromast HCs of zebraﬁsh were stained by DASPEI as described

previously³⁰to demonstrate the protection of the conjugates and NPs.

The conjugates were dissolved in DMSO. Then conjugates and NP

solutions were diluted with EM. The 5−6 dpf zebraﬁsh larvae were

pretreated with NAC (25 μM), DEX (90 μM), SAL (90 μM), the

DEX/SAL mixture (90 μM), conjugate 1 (80 μM), 2 (90 μM),

conjugate 1 NPs (80 μM), or 2 NPs (90 μM) for 1 h, and then except

for the control group, other experimental groups were exposed to

CDDP (700 μM) for 4 h. NAC was used as a positive control. To

evaluate mitochondrial damage within the HCs, the ﬂuorescent dye

DASPEI was applied to wild-type larvae. After being exposed to

CDDP, larvae were incubated in EM containing 0.005% DASPEI for

15 min and then washed three times with EM. Zebraﬁsh larvae were

anesthetized using tricaine mesylate (40 μg/mL, adjusted using EM)

for 5 min. The DASPEI-stained areas of HCs within SO1, SO2, O1,

and OC1 neuromasts were examined under a ﬂuorescence micro-

scope and measured using ImageJ.

Zebraﬁsh Embryo Toxicity Analysis. The Organization for

Economic Co-operation and Development (OECD) guidelines for

testing chemical toxicity were adapted for this study of the toxicity of

conjugates and NPs.⁶⁷EM (control) was used to dilute conjugate 1

(80 μM), 2 (90 μM), conjugate 1 NPs (80 μM), or 2 NPs (90 μM) in

24-well plates, and these were then exposed to 1.5 hpf zebraﬁsh

embryos (20 embryos per group). Microscopy was used to assess the

embryonic development 4, 24, 48, 72, 96, and 120 hpf. Additionally,

embryonic hatching, survival and malformation rates, the body length,

and the mean ﬂuorescence area of neuromasts HCs were quantiﬁed.

The malformation rate (percent) equals the number of malformed

embryos divided by the number of all embryos (the number of

malformed embryos includes all types of malformation and mortality).

The body length was measured as the distance from the center of an

eye to the tip of the tailbud.⁶⁸

Animal Drug Administration. A guinea pig weighing approx-

imately 300 g was provided by the Experimental Animal Center at

Southern Medical University (Guangzhou, China) and given free

access to food and water throughout the study. The CDDP-induced

hearing loss model was obtained as previously described.⁶⁴The

guinea pigs were randomly assigned to 10 groups with 12 animals per

group: (1) control, (2) CDDP, (3) positive control, (4) CDDP and

SAL, (5) CDDP and DEX, (6) CDDP and DEX/SAL mixture, (7)

CDDP and conjugate 1, (8) CDDP and conjugate 2, (9) CDDP and

conjugate 1 NPs, and (10) CDDP and conjugate 2 NPs. In the

meantime, DEX, the DEX/SAL mixture, conjugate 1, and conjugate 2

were dissolved in an aqueous solution with 1% Tween 80. Brieﬂy, the

control was given a transtympanic injection of saline. A dose of 12 mg

of CDDP/kg was administered by intraperitoneal injection. DEX,

SAL, the DEX/SAL mixture, conjugate 1, conjugate 2, and NPs were

3127

https://dx.doi.org/10.1021/acs.jmedchem.0c01916

J. Med. Chem. 2021, 64, 3115−3130

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Biocompatibility Assay. The experiment was set up as ﬁve

groups of guinea pigs, including control, conjugate 1, 2, and the

corresponding NPs. The control was subjected to transtympanic

injection of saline, and the other groups received transtympanic

administration at a dose of 0.23 mg/kg for two consecutive days. The

auditory function was measured by ABR recording after 3 days, and

then the animals were euthanized as described above. The cochleae

were decalciﬁed in 4% sodium ethylenediaminetetraacetic acid for 48

h, and the OC, SL, RM, SV, TM, and SGCs were dissected. After

dehydration and paraﬃn embedding, blocks were positioned parallel

to the cochlear axis and serial 5 μm sections were prepared. Sections

were then stained with hematoxylin and eosin; a light microscope was

used to observe the basic structure, and images were obtained with a

DMI 3000 camera.

ABBREVIATIONS

■

CDDP, cisplatin; HCs, hair cells; DEX, dexamethasone; SAL,

salvianolic acid B; GCs, glucocorticoids; GRs, glucocorticoid

receptors; EDCI, 1-ethyl-3-[3-(dimethylamino)propyl]-

carbodiimide hydrochloride; HOBt, 1-hydroxybenzotriazole;

DMAP, 4-(dimethylamino)pyridine; DIC, N,N′-diisopropyl-

carbodiimide; TFA, triﬂuoroacetic acid; DMF, dimethylforma-

mide; DMSO, dimethyl sulfoxide; HPLC, high-performance

liquid chromatography; NPs, nanoparticles; DMEM, Dulbec-

co’s modiﬁed Eagle’s medium; DLS, dynamic light scattering;

PBS, phosphate-buﬀered saline; TEM, transmission electron

microscopy; PDI, polydispersity index; HEI-OC1, House Ear

Institute-Organ of Corti 1; BSA, bovine serum albumin; NAC,

N-acetyl-^L-cysteine; DASPEI, 2-[4-(dimethylamino)styryl]-N-

ethylpyridinium iodide; MTT, 3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide; FBS, fetal bovine serum;

hpf, hours postfertilization; EM, embryo medium; dpf, days

postfertilization; SO1 and SO2, supraorbital; O1, otic; OC1,

occipital; SD, standard deviation; SEM, scanning electron

microscopy; SPL, sound pressure level; OHCs, outer hair cells;

IHCs, inner hair cells; SL&SV, spiral ligament and stria

vascularis; OC, organ of Corti; RM, Reissner’s membrane;

TM, tectorial membrane; SGCs, spiral ganglion cells

Statistical Analysis. All data are presented as means SD unless

otherwise stated. Statistical signiﬁcance was analyzed by one-way

analysis of variance using IBM SPSS 22.0, and p < 0.05 was

considered statistically signiﬁcant.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01916.

■

sı

Molecular formula strings (CSV)

Additional NMR spectra, HPLC spectra, and HRMS

data for compounds (PDF)

REFERENCES

■

(1) Hou, S. S.; Yang, Y.; Zhou, S.; Kuang, X.; Yang, Y. X.; Gao, H.;

Wang, Z. J.; Liu, H. Z. Novel SS-31 Modified Liposomes for

Improved Protective Efficacy of Minocycline against Drug-Induced

Hearing Loss. Biomater. Sci. 2018, 6, 1627−1635.

(2) Ostroumova, O. D.; Chikh, E. V.; Rebrova, E. V.; Ryazanova, A.

Yu.; Pereverzev, A. P. Drug-Induced Hearing Loss as a Manifestation

of Drug-Induced Ototoxicity. Vestn. Otorinolaringol. 2019, 84, 72−80.

(3) Laurell, G. Pharmacological Intervention in the Field of

Ototoxicity. HNO 2019, 67, 434−439.

(4) Dasari, S.; Tchounwou, P. B. Cisplatin in Cancer Therapy:

Molecular Mechanisms of Action. Eur. J. Pharmacol. 2014, 740, 364−

378.

(5) Fetoni, A. R.; Paciello, F.; Mezzogori, D.; Rolesi, R.; Eramo, S. L.

M.; Paludetti, G.; Troiani, D. Molecular Targets for Anticancer Redox

Chemotherapy and Cisplatin-Induced Ototoxicity: The Role of

Curcumin on PSTAT3 and Nrf-2 Signalling. Br. J. Cancer 2015,

113, 1434−1444.

AUTHOR INFORMATION

Corresponding Authors

■

Lu Wen − School of Pharmacy, Guangdong Pharmaceutical

University, Guangzhou 510006, China; Email: gywenl@

163.com

Chuan Bai − Institute of Human Virology, Sun Yat-sen

University, Guangzhou 510080, China; Email: baichuan@

mail.sysu.edu.cn

Gang Chen − School of Pharmacy, Guangdong Provincial Key

Laboratory of Advanced Drug Delivery, and Guangdong

Provincial Engineering Center of Topical Precise Drug

Delivery System, Guangdong Pharmaceutical University,

Guangzhou 510006, China; orcid.org/0000-0001-8369-

3006; Email: cg753@126.com

(6) Casares, C.; Ramírez-Camacho, R.; Trinidad, A.; Roldán, A.;

Jorge, E.; García-Berrocal, J. R. Reactive Oxygen Species in Apoptosis

Induced by Cisplatin: Review of Physiopathological Mechanisms in

Animal Models. Eur. Arch. Otorhinolaryngol. 2012, 269, 2455−2459.

(7) Campbell, K. C. M.; Le Prell, C. G. Drug-Induced Ototoxicity:

Diagnosis and Monitoring. Drug Saf. 2018, 41, 451−464.

(8) Zheng, Z.; Wang, Y. F.; Yu, H. Q.; Li, W.; Wu, J. F.; Cai, C. F.;

He, Y. Z. Salvianolic Acid B Inhibits Ototoxic Drug-Induced

Ototoxicity by Suppression of the Mitochondrial Apoptosis Pathway.

J. Cell. Mol. Med. 2020, 24, 6883−6897.

Authors

Ruiqin Ye − School of Pharmacy, Guangdong Pharmaceutical

University, Guangzhou 510006, China

Lifang Sun − School of Pharmacy, Guangdong Pharmaceutical

University, Guangzhou 510006, China

Jinghui Peng − School of Pharmacy, Guangdong

Pharmaceutical University, Guangzhou 510006, China

Aixin Wu − School of Pharmacy, Guangdong Pharmaceutical

University, Guangzhou 510006, China

(9) U.S. National Library of Medicine. https://www.clinicaltrials.gov

(accessed 2021-01-12).

Xiaozhu Chen − School of Pharmacy, Guangdong

Pharmaceutical University, Guangzhou 510006, China

(10) Yu, D. H.; Gu, J. Y.; Chen, Y. M.; Kang, W.; Wang, X. L.; Wu,

H. Current Strategies to Combat Cisplatin-Induced Ototoxicity.

Front. Pharmacol. 2020, 11, 999.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01916

(11) Fernandez, K. A.; Watabe, T.; Tong, M.; Meng, X.; Tani, K.;

Kujawa, S. G.; Edge, A. S. Trk Agonist Drugs Rescue Noise-Induced

Hidden Hearing Loss. JCI Insight 2020, 29, 142572.

(12) Ingersoll, M. A.; Malloy, E. A.; Caster, L. E.; Holland, E. M.;

Xu, Z.; Zallocchi, M.; Currier, D.; Liu, H.; He, D. Z. Z.; Min, J.; Chen,

T.; Zuo, J.; Teitz, T. BRAF Inhibition Protects against Hearing Loss

in Mice. Sci. Adv. 2020, 6, No. eabd0561.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

The authors gratefully acknowledge the ﬁnancial support from

the National Natural Science Foundation of China (Grants

81773917 and 82074028).

(13) El Sabbagh, N. G.; Sewitch, M. J.; Bezdjian, A.; Daniel, S. J.

Intratympanic Dexamethasone in Sudden Sensorineural Hearing

3128

https://dx.doi.org/10.1021/acs.jmedchem.0c01916

J. Med. Chem. 2021, 64, 3115−3130

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Loss: A Systematic Review and Meta-Analysis. Laryngoscope 2017,

127, 1897−1908.

(31) Katary, M. A.; Abdelsayed, R.; Alhashim, A.; Abdelhasib, M.;

Elmarakby, A. A. Salvianolic Acid B Slows the Progression of Breast

Cancer Cell Growth via Enhancement of Apoptosis and Reduction of

Oxidative Stress, Inflammation, and Angiogenesis. Int. J. Mol. Sci.

2019, 20, 5653.

(32) Zheng, Z.; Wang, Y.; Yu, H.; Li, W.; Wu, J. F.; Cai, C. F.; He, Y.

Z. Salvianolic Acid B Inhibits Ototoxic Drug-Induced Ototoxicity by

Suppression of the Mitochondrial Apoptosis Pathway. J. Cell. Mol.

Med. 2020, 24, 6883−6897.

(33) Zhou, L.; Chow, M. S. S.; Zuo, Z. Effect of Sodium Caprate on

the Oral Absorptions of Danshensu and Salvianolic Acid B. Int. J.

Pharm. 2009, 379, 109−118.

(34) Li, Y.; Yang, D. D.; Zhu, C. Y. Impact of Sodium N-[8-(2-

Hydroxybenzoyl)amino]-Caprylate on Intestinal Permeability for

Notoginsenoside R1 and Salvianolic Acids in Caco-2 Cells Transport

and Rat Pharmacokinetics. Molecules 2018, 23, 2990.

(35) Xu, Z.; Hou, M.; Shi, X.; Gao, Y. E.; Xue, P.; Liu, S.; Kang, Y. J.

Rapidly Cell-Penetrating and Reductive Milieu-Responsive Nano-

aggregates Assembled from an Amphiphilic Folate-Camptothecin

Prodrug for Enhanced Drug Delivery and Controlled Release.

Biomater. Sci. 2017, 5, 444−454.

(36) Hou, M.; Xue, P.; Gao, Y. E.; Ma, X. Q.; Bai, S.; Kang, Y. J.; Xu,

Z. G. Gemcitabine-Camptothecin Conjugates: A Hybrid Prodrug for

Controlled Drug Release and Synergistic Therapeutics. Biomater. Sci.

2017, 5, 1889−1897.

(37) Cheng, Y.; Ji, Y. H. Mitochondria-Targeting Nanomedicine

Self-Assembled from GSH-Responsive Paclitaxel-SS-Berberine Con-

jugate for Synergetic Cancer Treatment with Enhanced Cytotoxicity.

J. Controlled Release 2020, 318, 38−49.

(14) Marshak, T.; Steiner, M.; Kaminer, M.; Levy, L.; Shupak, A.

Prevention of Cisplatin-Induced Hearing Loss by Intratympanic

Dexamethasone. Otolaryngol.–Head Neck Surg. 2014, 150, 983−990.

(15) Lee, S. H.; Lyu, A. R.; Shin, S. A.; Jeong, S. H.; Lee, S. A.; Park,

M. J.; Park, Y. H. Cochlear Glucocorticoid Receptor and Serum

Corticosterone Expression in a Rodent Model of Noise-Induced

Hearing Loss: Comparison of Timing of Dexamethasone Admin-

istration. Sci. Rep. 2019, 9, 12646.

(16) Pujols, L.; Alobid, I.; Benítez, P.; Martínez-Antón, A.; Roca-

Ferrer, J.; Fokkens, W. J.; Mullol, J.; Picado, C. Regulation of

Glucocorticoid Receptor in Nasal Polyps by Systemic and Intranasal

Glucocorticoids. Allergy 2008, 63, 1377−1386.

(17) Shimazaki, T.; Ichimiya, I.; Suzuki, M.; Mogi, G. Localization of

Glucocorticoid Receptors in the Murine Inner Ear. Ann. Otol., Rhinol.,

Laryngol. 2002, 111, 1133−1138.

(18) Terakado, M.; Kumagami, H.; Takahashi, H. Distribution of

Glucocorticoid Receptors and 11 Beta-Hydroxysteroid Dehydrogen-

ase Isoforms in the Rat Inner Ear. Hear. Res. 2011, 280, 148−156.

(19) Tahera, Y.; Meltser, I.; Johansson, P.; Bian, Z.; Stierna, P.;

Hansson, A. C.; Canlon, B. NF-kappaB Mediated Glucocorticoid

Response in the Inner Ear after Acoustic Trauma. J. Neurosci. Res.

2006, 83, 1066−1076.

(20) Wang, X. L.; Chen, Y. M.; Tao, Y.; Gao, Y. G.; Yu, D. H.; Wu,

H. A666-Conjugated Vanoparticles Target Prestin of Quter Hair Cells

Preventing Cisplatin-Induced Hearing Loss. Int. J. Nanomed. 2018, 13,

7517−7531.

(21) Herr, I.; Ucur, E.; Herzer, K.; Okouoyo, S.; Ridder, R.;

Krammer, P. H.; Doeberitz, M. V. K.; Debatin, K. M. Glucocorticoid

Cotreatment Induces Apoptosis Resistance toward Cancer Therapy in

Carcinomas. Cancer Res. 2003, 63, 3112−3120.

(38) Zheng, X. H.; Li, Z. S.; Chen, L.; Xie, Z. G.; Jing, X. B. Self-

Assembly of Porphyrin-Paclitaxel Conjugates into Nanomedicines:

Enhanced Cytotoxicity due to Endosomal Escape. Chem. - Asian J.

2016, 11, 1780−1784.

̈

(22) Ozel, H. E.; Ozdogan, F.; Gu

̧

̈

rgen, S. G.; Esen, E.; Genc,̧ S.;

Selcuk, A. Comparison of the Protective Effects of Intratympanic

Dexamethasone and Methylprednisolone against Cisplatin-Induced

Ototoxicity. J. Laryngol. Otol. 2016, 130, 225−234.

(39) Wang, Y.; Huang, P.; Hu, M.; Huang, W.; Zhu, X. Y.; Yan, D. Y.

Self-Delivery Nanoparticles of Amphiphilic Methotrexate-Gemcita-

bine Prodrug for Synergistic Combination Chemotherapy via Effect of

Deoxyribonucleotide Pools. Bioconjugate Chem. 2016, 27, 2722−2733.

(40) Coyne, C P; Narayanan, L. Dexamethasone-(C₂₁-phosphor-

amide)-[anti-EGFR]: Molecular Design, Synthetic Organic Chemistry

Reactions, and Antineoplastic Cytotoxic Potency against Pulmonary

Adenocarcinoma (A549). Drug Des., Dev. Ther. 2016, 10, 2575−2597.

(41) Kuang, H. Z.; Wang, Y.; Hu, J. F.; Wang, C. S.; Lu, S. Y.; Mo, X.

M. A Method for Preparation of an Internal Layer of Artificial

Vascular Graft Co-Modified with Salvianolic Acid B and Heparin.

ACS Appl. Mater. Interfaces 2018, 10, 19365−19372.

(42) Wu, D.; Chen, Y.; Wen, S.; Wen, Y.; Wang, R.; Zhang, Q. T.;

Qin, G.; Yi, H. M.; Wu, M.; Lu, L.; Tao, X. J.; Deng, X. Y.

Synergistically Enhanced Inhibitory Effects of Pullulan Nanoparticle-

Mediated Co-Delivery of Lovastatin and Doxorubicin to Triple-

Negative Breast Cancer Cells. Nanoscale Res. Lett. 2019, 14, 314.

(43) Young, J. J.; Chen, C. C.; Chen, Y. C.; Cheng, K. M.; Yen, H. J.;

Huang, Y. C.; Tsai, T. N. Positively and Negatively Surface-Charged

Chondroitin Sulfate-Trimethylchitosan Nanoparticles as Protein

Carriers. Carbohydr. Polym. 2016, 137, 532−540.

(44) Li, Y.; Lin, J. Y.; Ma, J. Y.; Song, L.; Lin, H. R.; Tang, B. W.;

Chen, D. Y.; Su, G. H.; Ye, S. F.; Zhu, X.; Luo, F. H.; Hou, Z. Q.

Methotrexate-Camptothecin Prodrug Nanoassemblies as a Versatile

Nanoplatform for Biomodal Imaging-Guided Self-Active Targeted

and Synergistic Chemotherapy. ACS Appl. Mater. Interfaces 2017, 9,

34650−34665.

(45) Upendar, S.; Mani, E.; Basavaraj, M. G. Aggregation and

Sstabilization of Colloidal Spheroids by Oppositely Charged Spherical

Nanoparticles. Langmuir 2018, 34, 6511−6521.

(23) Wikström, A. C.; Bakke, O.; Okret, S.; Brönnegård, M.;

Gustafsson, J. A. Intracellular Localization of the Glucocorticoid

Receptor: Evidence for Cytoplasmic and Nuclear Localization.

Endocrinology 1987, 120, 1232−1242.

(24) Ko, Y. S.; Jin, H.; Park, S. W.; Kim, H. J. Salvianolic Acid B

Protects against OxLDL-Induced Endothelial Dysfunction under

High-Glucose Conditions by Downregulating ROCK1-Mediated

Mitophagy and Apoptosis. Biochem. Pharmacol. 2020, 174, 113815.

(25) Li, C. L.; Liu, B.; Wang, Z. Y.; Xie, F.; Qiao, W.; Cheng, J.;

Kuang, J. Y.; Wang, Y.; Zhang, M. X.; Liu, D. S. Salvianolic Acid B

Improves Myocardial Function in Diabetic Cardiomyopathy by

Suppressing IGFBP3. J. Mol. Cell. Cardiol. 2020, 139, 98−112.

(26) Shu, T.; Liu, C.; Pang, M.; He, L.; Yang, B.; Fan, L.; Zhang, S.

F.; Wang, X.; Liu, B.; Rong, L. M. Salvianolic Acid B Promotes Neural

Differentiation of Induced Pluripotent Stem Cells via PI3K/AKT/

GSK3 β /β -Catenin Pathway. Neurosci. Lett. 2018, 671, 154−160.

(27) Qin, C. Z.; Ren, X.; Zhou, H. H.; Mao, X. Y.; Liu, Z. Q.

Inhibitory Effect of Salvianolate on Human Cytochrome P450 3A4 in

Vitro Involving a Noncompetitive Manner. Int. J. Clin. Exp. Med.

2015, 8, 15549−15555.

(28) Feng, X. J.; Li, Y.; Wang, Y. N.; Li, L. L.; Little, P. J.; Xu, S. W.;

Liu, S. Danhong Injection in Cardiovascular and Cerebrovascular

Diseases: Pharmacological Actions, Molecular Mechanisms, and

Therapeutic Potential. Pharmacol. Res. 2019, 139, 62−75.

(29) Shi, Y. N.; Pan, D.; Yan, L. H.; Chen, H. Y.; Zhang, X. M.;

Yuan, J. H.; Mu, B. Salvianolic Acid B Improved Insulin Resistance

through Suppression of Hepatic ER Stress in Ob/ob Mice. Biochem.

Biophys. Res. Commun. 2020, 526, 733−737.

(46) Rodriguez de Anda, D. A.; Ohannesian, N.; Martirosyan, K. S.;

Chew, S. A. Effects of Solvent Used for Fabrication on Drug Loading

and Release Kinetics of Electrosprayed Temozolomide-Loaded PLGA

Microparticles for the Treatment of Glioblastoma. J. Biomed. Mater.

Res., Part B 2019, 107, 2317−2324.

(30) Wu, A. X.; Li, J.; Chen, G.; Wen, L. Protective Effect of Salvia

Miltiorrhiza Extract and Salvianolic Acid B against Gentamicin-

Induced Ototoxicity in Vitro and in Vivo. Guangdong Yaoke Daxue

Xuebao 2019, 35, 76−81.

3129

https://dx.doi.org/10.1021/acs.jmedchem.0c01916

J. Med. Chem. 2021, 64, 3115−3130

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(47) Albuquerque, A. A.; Rossato, M.; de Oliveira, J. A. A.;

Hyppolito, M. A. Understanding the Anatomy of Ears from Guinea

Pigs and Rats and Its Use in Basic Otologic Research. Brazilian

Journal of Otorhinolaryngology 2009, 75, 43−49.

(67) Sobanska, M.; Scholz, S.; Nyman, A. M.; Cesnaitis, R.;

Gutierrez Alonso, S.; Kluver, N.; Kuhne, R.; Tyle, H.; de Knecht, J.;

̈

Dang, Z.; Lundbergh, I.; Carlon, C.; De Coen, W. Applicability of the

Fish Embryo Acute Toxicity (FET) Test (OECD 236) in the

Regulatory Context of Registration, Evaluation, Authorisation, and

Restriction of Chemicals (REACH). Environ. Toxicol. Chem. 2018, 37,

657−670.

(68) Liu, W.; Huang, G.; Su, X. Y.; Li, S. Y.; Wang, Q.; Zhao, Y. Y.;

Liu, Y.; Luo, J. S.; Li, Y.; Li, C. W.; Yuan, D. S.; Hong, H. H.; Chen, X.

J.; Chen, T. K. Zebrafish: A Promising Model for Evaluating the

Toxicity of Carbon Dot-Based Nanomaterials. ACS Appl. Mater.

Interfaces 2020, 12, 49012−49020.

(48) Creber, N. J.; Eastwood, H. T.; Hampson, A. J.; Tan, J.;

O’Leary, S. J. A Comparison of Cochlear Distribution and

Glucocorticoid Receptor Activation in Local and Systemic Dexa-

methasone Drug Delivery Regimes. Hear. Res. 2018, 368, 75−85.

(49) Chen, G.; Zhang, X.; Yang, F.; Mu, L. Disposition of

Nanoparticle-Based Delivery System via Inner Ear Administration.

Curr. Drug Metab. 2010, 11, 886−897.

(50) Roehm, P.; Hoffer, M.; Balaban, C. D. Gentamicin Uptake in

the Chinchilla Inner Ear. Hear. Res. 2007, 230, 43−52.

(51) Shih, C. P.; Chen, H. C.; Lin, Y. C.; Chen, H. K.; Wang, H.;

Kuo, C. Y.; Lin, Y. Y.; Wang, C. H. Middle-Ear Dexamethasone

Delivery via Ultrasound Microbubbles Attenuates Noise-Induced

Hearing Loss. Laryngoscope 2019, 129, 1907−1914.

(52) Canlon, B.; Meltser, I.; Johansson, P.; Tahera, Y. Glucocorti-

coid Receptors Modulate Auditory Sensitivity to Acoustic Trauma.

Hear. Res. 2007, 226, 61−69.

(53) Heinrich, U. R.; Strieth, S.; Schmidtmann, I.; Stauber, R.;

Helling, K. Dexamethasone Prevents Hearing Loss by Restoring

Glucocorticoid Receptor Expression in the Guinea Pig Cochlea.

Laryngoscope 2016, 126, E29−E34.

(54) Trune, D. R.; Kempton, J. B. Aldosterone and Prednisolone

Control of Cochlear Function in MRL/MpJ-Fas(lpr) Autoimmune.

Hear. Res. 2001, 155, 9−20.

(55) Trune, D. R.; Kempton, J. B.; Gross, N. D. Mineralocorticoid

Receptor Mediates Glucocorticoid Treatment Effects in the Auto-

immune Mouse Ear. Hear. Res. 2006, 212, 22−32.

(56) Kadhim, H. J.; Kang, S. W.; Kuenzel, W. J. Differential and

Temporal Expression of Corticotropin Releasing Hormone and Its

Receptors in the Nucleus of the Hippocampal Commissure and

Paraventricular Nucleus during the Stress Response in Chickens

(Gallus gallus ). Brain Res. 2019, 1714, 1−7.

(57) Li, M.; Ai, M. Y.; Yang, Y. Z.; Yao, X. Y.; Zhou, Z. M.; Wang, H.

Y.; Li, C.; Xu, K. X. Silk-Coated Dexamethasone Non-Spherical

Microcrystals for Local Drug Delivery to Inner Ear. Eur. J. Pharm. Sci.

2020, 150, 105336.

(58) Rarey, K. E.; Curtis, L. M. Receptors for Glucocorticoids in the

Human Inner Ear. Otolaryngol.–Head Neck Surg. 1996, 115, 38−41.

(59) Borski, R. J. Nongenomic Membrane Actions of Glucocorti-

coids in Vertebrates. Trends Endocrinol. Metab. 2000, 11, 427−436.

(60) Hirose, Y.; Tabuchi, K.; Oikawa, K.; Murashita, H.; Sakai, S.;

Hara, A. The Effects of the Glucocorticoid Receptor Antagonist

RU486 and Phospholipase A2 Inhibitor Quinacrine on Acoustic

Injury of the Mouse Cochlea. Neurosci. Lett. 2007, 413, 63−67.

(61) Chen, Y. S.; Tseng, F. Y.; Liu, T. C.; Lin-Shiau, S. Y.; Hsu, C. J.

Involvement of Nitricoxide Generation in Noise-Induced Temporary

Threshold Shift in Guinea Pigs. Hear. Res. 2005, 203, 94−100.

(62) Kalinec, G.; Thein, P.; Park, C.; Kalinec, F. HEI-OC1 Cells as a

Model for Investigating Drug Cytotoxicity. Hear. Res. 2016, 335,

105−117.

(63) Li, J.; Wu, G. M.; Qin, C.; Chen, W.; Chen, G.; Wen, L.

Structure Characterization and Otoprotective Effects of a New

Endophytic Exopolysaccharide from Saffron. Molecules 2019, 24, 749.

(64) Spankovich, C.; Lobarinas, E.; Ding, D.; Salvi, R.; Le Prell, C.

G. Assessment of Thermal Treatment via Irrigation of External Ear to

Reduce Cisplatin-Induced Hearing Loss. Hear. Res. 2016, 332, 55−60.

(65) Pang, J. Q.; Xiong, H.; Yang, H. D.; Ou, Y. K.; Xu, Y. D.;

Huang, Q. H.; Lai, L.; Chen, S. J.; Zhang, Z. G.; Cai, Y. X.; Zheng, Y.

Q. Circulating MiR-34a Levels Correlate with Age-Related Hearing

Loss in Mice and Humans. Exp. Gerontol. 2016, 76, 58−67.

(66) Zhang, L. P.; Xu, Y.; Cao, W. J.; Xie, S. B.; Wen, L.; Chen, G.

Understanding the Translocation Mechanism of PLGA Nanoparticles

Across Round Window Membrane into the Inner Ear: A Guideline for

Inner Ear Drug Delivery Based on Nanomedicine. Int. J. Nanomed.

2018, 13, 479−492.

3130

https://dx.doi.org/10.1021/acs.jmedchem.0c01916

J. Med. Chem. 2021, 64, 3115−3130

Article Doi

Design, Synthesis, and Biological Evaluation of Dexamethasone-Salvianolic Acid B Conjugates and Nanodrug Delivery against Cisplatin-Induced Hearing Loss

DOI: 10.1021/acs.jmedchem.0c01916

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acs.jmedchem.0c01916

Full text of DOI:10.1021/acs.jmedchem.0c01916

Products guided by the article

R&D Labs maybe for 37927-01-8

Relevant to this article

Hot Product