Stepwise dual targeting and dual responsive polymer micelles for mitochondrion therapy

DOI: 10.1016/j.jconrel.2020.03.011

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Journal of Controlled Release p. 157 - 169 (2020)

Update date:2022-08-28

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Authors:

Wang, Yi

Wei, Guoqing

Yang, Guang

Zhang, Xiaobin

Zhao, Jingya

Zhou, Shaobing

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Article abstract of DOI:10.1016/j.jconrel.2020.03.011

Methods to selectively destroy mitochondria of tumor cells and induce cell apoptosis with nanomedicine constitute challenges in cancer therapy. In the present study, we develop cell membrane/mitochondria dual targeting and pH/redox dual responsive nanoparticles for mitochondrion therapy. The nanoparticles are fabricated by the self-assembly of triphenylphosphonium (TPP) grafted poly(ethylene glycol)(PEG)-poly(d,L-lactide)(PLA) copolymers (TPP-PEG-ss-PLA) using disulfide bonds as the intermediate linkers. To shield the surface positive charge of the nanoparticles from TPP composition, chondroitin sulfate (CS) is employed to coat the nanoparticles, and this prolongs blood circulation while endowing an active targeting ability to the cell membrane. In acidic lyso-somes/endosomes, the negatively charged CS layer falls away to expose the TPP component. Subsequently, in the cyto-plasm, the nanoparticles can anchor to the mitochondrial outer membrane by TPP-mediated targeting, thereby inducing a decrease in the membrane potential and opening of the permeability transition pore. Thus, the overproduction of ROS in the mitochondria promotes cell apoptosis. The released DOX directly diffuse into the mitochondria, thereby resulting in mito-chondrial DNA damage. Therefore, the nanoparticles exhibit significant potential in terms of a new avenue for mitochondrion therapy in cancer treatment.

Full text of DOI:10.1016/j.jconrel.2020.03.011

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Stepwise dual targeting and dual responsive polymer micelles for

mitochondrion therapy

Xiaobin Zhang, Yi Wang, Guoqing Wei, Jingya Zhao, Guang

Yang, Shaobing Zhou

PII:

S0168-3659(20)30163-2

DOI:

https://doi.org/10.1016/j.jconrel.2020.03.011

COREL 10213

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Journal of Controlled Release

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Revised date:

Accepted date:

30 October 2019

24 February 2020

7 March 2020

Please cite this article as: X. Zhang, Y. Wang, G. Wei, et al., Stepwise dual targeting

and dual responsive polymer micelles for mitochondrion therapy, Journal of Controlled

Release (2019), https://doi.org/10.1016/j.jconrel.2020.03.011

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Stepwise dual targeting and dual responsive polymer micelles for

mitochondrion therapy

Xiaobin Zhang^a,b,c‡, Yi Wang^a‡, Guoqing Wei^a, Jingya Zhao^a, Guang Yang^aand Shaobing

Zhou^a*

^aKey Laboratory of Advanced Technologies of Materials, Ministry of Education, School of

Materials Science and Engineering, Southwest Jiaotong University, Chengdu, Sichuan

610031, P.R. China

^bRadiation Chemistry Department, Sichuan Institute of Atomic Energy, Chengdu, Sichuan

610101, P.R. China

^cIrradiation Preservation Key Laboratory of Sichuan Province, Chengdu, Sichuan 610101,

P.R. China

* Corresponding author.

E-mail address: shaobingzhou@swjtu.edu.cn (S. Z.).

CRediT author statement

Xiaobin Zhang: Conceptualization, Methodology, Software Xiaobin Zhang,

Yi Wang: Data curation, Investigation, Writing- Original draft preparation.

Guoqing Wei: Visualization, Investigation. Jingya Zhao, Guang Yang:

Investigation and Editing Shaobing Zhou:Writing- Reviewing and Editing

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Abstracts

Methods to selectively destroy mitochondria of tumor cells and induce cell apoptosis with

nanomedicine constitute challenges in cancer therapy. In the present study, we develop cell

membrane/mitochondria dual targeting and pH/redox dual responsive nanoparticles for

mitochondrion therapy. The nanoparticles are fabricated by the self-assembly of

triphenylphosphonium (TPP) grafted poly(ethylene glycol)(PEG)-poly(d,l-lactide)(PLA)

copolymers (TPP-PEG-ss-PLA) using disulfide bonds as the intermediate linkers. To shield

the surface positive charge of the nanoparticles from TPP composition, chondroitin sulfate

(CS) is employed to coat the nanoparticles, and this prolongs blood circulation while

endowing an active targeting ability to the cell membrane. In acidic lyso-somes/endosomes,

the negatively charged CS layer falls away to expose the TPP component. Subsequently, in the

cyto-plasm, the nanoparticles can anchor to the mitochondrial outer membrane by TPP-

mediated targeting, thereby inducing a decrease in the membrane potential and opening of the

permeability transition pore. Thus, the overproduction of ROS in the mitochondria promotes

cell apoptosis. The released DOX directly diffuse into the mitochondria, thereby resulting in

mito-chondrial DNA damage. Therefore, the nanoparticles exhibit significant potential in

terms of a new avenue for mitochondrion therapy in cancer treatment.

KEYWORDS: Mitochondrion therapy, dual targeting, micelle, drug delivery, nanoparticles

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1. Introduction

A popular concept involves engineering nanoparticles to improve anticancer drug delivery

and subsequent chemotherapy to malignant tumor. To achieve optimal delivery efficiency,

nanoparticles should exhibit functions such as therapeutic targeting for cancerous

organization, cell membranes, and subcellular organelles. The targeted delivery of a

therapeutic agent to cancer tissues and cell membranes with nanoparticles is widely

investigated [1-4].After the nanoparticles enter the cell, it is important for the carried cargos

to reach specific organelles. The specific delivery of a therapeutic agent to molecular targets

associated with certain organelles significantly improves drug action efficacy [5-8]. Therefore,

increased research attention focuses on the organelle-specific targeting delivery of

nanoparticles [9-11].

In a cell, the organelles mainly include nucleus, mitochondria, lysosomes, and ribosome.

Specifically, mitochondria are the cell’s powerhouse and maintain pivotal physiological

functions [12-14], and also exert a lethal function via controlling the activation of an apoptotic

effector in cancer cells [14, 15]. Additionally, the mitochondria also contains DNA. However,

in contrast to nuclear DNA, introns are absent in mitochondrial DNA that only contains

essential genes for the expression of mitochondrial proteins, tRNA, and rRNA [16].

Additionally, in the mitochondria, the DNA repair pathways are not processed, and this is

different from that in the nucleus [17]. Thus, if the DNA-damaging anticancer drugs are

delivered to the mitochondrial DNA, it presents increased advantages when compared to the

method of targeting the nuclear genome.

However, targeting mitochondrial DNA is a process that is hard to achieve. The reason for

this is mainly because mitochondrial membrane exhibits a typical thickness in which the outer

and inner membranes have highly negative potentials (∆Ψ = -150 to -180 mV) [18, 91], and

the special hydrophobic and dense double-membrane structure acts as a formidable barrier to

prevent bioactive compounds from gaining entry [16]. The DNA-damaging anticancer drugs

cannot enter the mitochondria in which therapeutic effect is extremely limited. Therefore, to

achieve a high therapeutic effect of mitochondrial DNA-damaging anticancer drugs, first, the

drugs should precisely target mitochondria; and the permeability of mitochondrial membrane

should be subsequently enhanced to allow the drugs to enter the mitochondria.

To selectively deliver a drug to the mitochondria, mitochondria-targeting signal peptides

(MTSs) [20, 21], oligoguanidinium [22], and triphenylphosphonium (TPP) [23, 24] are widely

employed to modify drug carriers in drug delivery systems. The ability to target mitochondria

significantly improves the drug delivery efficacy of macromolecular carriers and widens the

application of anticancer drugs [15]. Among these moieties for mitochondria targeting, TPP as

a cation exhibits a unique chemical structure with lipophilicity and delocalized positive

charge. It is selectively targeted to the mitochondria based on mitochondrial-membrane-

potential-driven accumulation through the lipophilic phenyl groups and cationic phosphonium

of TPP due to the negative potential gradient of the mitochondrion [15, 25].

To enhance the permeability of mitochondrial membrane and allow the therapeutic agents

to enter mitochondria, a feasible method involves destroying the function of mitochondrial

membrane. The damage to the mitochondria membrane affects the components of the

mitochondrial respiratory chain and leads to the depolarization of mitochondria with a

decrease in the mitochondria membrane potential (∆Ψ), thereby resulting in the opening of the

permeability transition pore (PTP) of mitochondrial membrane and overproduction of the

reactive oxygen species (ROS) [26, 27]. The functions of TPP possessing both mitochondrial

targeting and mitochondrial damage are accepted [28-30]. Extant studies indicate that after

anchoring in mitochondrial outer membrane, TPP makes the mitochondrial permeability

transition, and this is also highly associated with the ∆Ψ [31]. However, the capacity to

enhance the permeability of mitochondrial outer membrane is rarely examined.

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As a concept of mitochondrion therapy, we synthesize a pH/redox dual responsive and cell

membrane-/mitochondria- dual targeting nanoparticle that exhibits a significant capacity to

inducing mitochondrial membrane depolarization to increase the permeability of

mitochondrial membrane and allow therapeutic agents to enter mitochondria to cause

mitochondrial DNA damage. As shown in Scheme 1A, the nanoparticles loaded with

anticancer drug doxorubicin (DOX) are fabricated by the self-assembly of TPP grafted

poly(ethylene glycol)(PEG)-poly(D, L-lactide) (PLA) copolymers using disulfide bonds as

the intermediate linker (TPP-PEG-ss-PLA). To prolong the blood circulation and increase the

endocytosis of tumor cells, the surface positive charges from TPP composition are

transformed into negative charges via coating the nanoparticles with chondroitin sulfate (CS)

(Scheme 1B). CD44 is a transmembrane glycoprotein which is overexpressed in most human

cancers, including hepatoma carcinoma, breast cancer, ovarian cancer, squamous cell and so

forth [32]. The CS layer as a CD44 acceptor also exhibits an ability to selectively mediate the

endocytosis of the nanoparticles [33]. This would result in selective and preferential drug

delivery to the tumor cells [34]. CS is hydrophilic and stable at pH 7.4 [35], it becomes

hydrophobic when the pH decreases to 5.5 due to dissociation of carboxyl group [36, 37]. In

lysosomes/endosomes, the negative charged CS layer is decoated upon decreasing pH from

7.4 to 5.5, then falls off to expose the TPP due to the protonation of CS in acidic

microenvironment, and surface charge reversal occurs on the nanoparticles. Subsequently, the

nanoparticles target and anchor into mitochondrial outer membrane, thereby inducing the

depolarization of mitochondrial membrane with decreases in the ∆Ψ and increases in the

mitochondria membrane permeability. Thus, the overproduction of ROS in mitochondria

promotes cells apoptosis (Scheme 1C), and the released DOX from the disassembly of

nanoparticles by glutathione (GSH) triggered redox-responsiveness directly diffuses into

mitochondria since the membrane permeability increases. This results in mitochondrial DNA

damage. Simultaneously, the other small part of the released DOX diffuses into nucleus,

thereby leading to nuclear DNA damage. All the aforementioned actions promote the

apoptosis of tumor cells.

Scheme 1. A) Formation of the micelles by the self-assembly of TPP-PEG-ss-PLA polymer and

subsequently coated with CS layer, and the disassembly of the nanoparticle via the initial deshielding of CS

layer by pH-response followed by the cleavage of the disulfide bonds by GSH response. B) Schematic

illustration of the stepwise dual targeting and dual response of the micelles to induce apoptosis of tumor

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cells. 1) Increased endocytosis through receptor-mediated cell membrane targeting; 2) Mitochondrial

targeting via TPP exposure due to pH response, which causes decreases in the membrane potential (ΔΨm)

and overproduction of the ROS and finally leads to cell apoptosis; 3) The DOX released due to the

disassembly by GSH response diffuses to the mitochondria and the nucleus and damages DNA. C)

Magnification of the series process including mitochondrial targeting, decreases in the membrane potential

(ΔΨm), overproduction of the ROS, and drug diffusion into the mitochondria.

2. Materials and Methods

2.1. Materials

Polyethylene glycol (PEG, Mw=2000) was purchased from Aldrich. D, L-lactide was

prepared in our laboratory. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

(EDC·HCl) , 4-(dimethylamino)pyridine (DMAP) and Carboxyl dextran (DEX, Mw=7000)

were purchased from Biokem Chemical Reagent (Chengdu, China). JC-1 and 2,7-dichloro

fluoresce in diacetate (DCFH-DA) were obtained from Aldrich Chemical Co.

Triphenylphosphine and glutathione (GSH) were purchased from Adamas-beta Reagent, Ltd.

(Shanghai, China). Bis(2-hydroxyethyl) Disulfide and Chondroitin sulfate sodium salt (CS,

Mn=1526.03Da) was obtained from TCI (Shanghai) Development Co. Ltd. All other

chemicals were purchased from Kelong Chemical Reagent Ltd.(Chengdu, China) and used

without any treatments.

2.2. Cell Lines and Culture Conditions

Liver hepatocellular carcinoma (HepG2) were purchased from the Cell Bank of China

Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 supplemented

with 10% FBS fetal bovine serum (FBS) at 37 °C in a 5% CO₂atmosphere and 100%

humidity. Endothelial cells (EC) were obtained from Sichuan University (China). EC Cells

were cultured in HyClone F12 supplemented with 10% fetal bovine serum (FBS).

2.3. Animals

Female Balb/c mice (18 ± 2 g) and female nude mice (20 ± 2 g) were feed at the condition

of 25 °C and 55% of humidity and approved by the Institutional Animal Care and Use

Committee of Sichuan University. All animal experiments were carried out in compliance

with guidelines.

2.4 Synthesis of Boc-NH2

14 mL Ethylenediamine is dissolved into 200 mL dichloromethane (DCM), and the mixture

is stirred at 0 °C to -5 °C. Additionally, 4.35 g (Boc)₂O solution is added into 100 mL DCM,

and the solution is then added dropwise to the reaction slowly within 6 h. It is reacted for

another 24 h, and the reaction mixture is then filtered. The filtrate is washed with saturated

salt water thrice, and the organic phase is separately concentrated under vacuum to yield a

yellowish liquid (Boc-NH₂, yield: 81%).¹H NMR (400 MHz, CDCl₃) δ 5.64–5.40 (br, 1H,

C(=O)-NH), 3.15–2.78 (m, 2H, NHCH₂CH₂, J = 5.8 Hz), 2.59 (t, J = 6.0 Hz, 2H, CH₂NH₂),

1.23 (s, 9H, t-Bu).

2.5. Synthesis of (3-Carboxypropyl)triphenylphosphonium bromide (TPP-COOH)

Triphenylphosphine (222 mg 0.16 mmol) and 4-Bromobutyric acid (70 mg 0.37 mmol) are

dissolved in 100 mL N,N-dimethyl formamide (DMF). The mixture is kept stirring at 110 °C

for 72 h, and the reaction mixture is then filtered to acquire white solid (TPP-COOH) (yield:

84%). H NMR (400 MHz, DMSO-d6) δ 12.35 (s, 1H), 8.02–7.56 (m, 15H), 3.60 (td, J =

13.5, 7.7 Hz, 2H), 2.56–2.36 (m, 2H), 1.70 (td, J = 11.7, 9.7, 5.9 Hz, 2H).

2.6. Synthesis of Boc-TPP

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Furthermore, Boc-NH₂(1 g, 6.25 mmol), TPP-COOH (0.549 g, 1.28 mmol) and NHS

(0.443 g, 3.85 mmol), and EDC·HCl (0.989 g, 5.16 mmol) are dissolved in 50 mL DMF, and

EDC·HCl (0.989 g, 5.16 mmol) is added while stirring. The mixture is stirred at room-

temperature until the Boc-NH₂is completely reacted via monitoring with thin layer

chromatography (TLC). When the reaction is complete, 5 mL deionized water is added and

stirred for 30 min to quench the reaction. The solvent is removed in vacuo, and the residue is

redissolved in 50 mL DCM and washed with 30 mL saturated salt water. The organic phase is

dried by using anhydrous magnesium sulfate. After filtering the solid, the filtrate is condensed

to obtain a pale yellow liquid (Boc-TPP, 73% yield).

2.7. Synthesis of COOH-PEG-COOH and Boc-NH-ss-NH₂

COOH-PEG-COOH and Boc-NH-ss-NH₂are synthesized according to previously reported

studies [38]. COOH-PEG-COOH: H NMR (400 MHz, CDCl₃) δ 4.26 (br, 4H, -

CH₂CH₂OCO-), 4.21–3.24 (m, 156H, -OCH₂CH₂O- and -C-(O)OCH₂CH₂-), 2.62 (br, 8H, -

CH₂CH₂C(O)OH). Boc-NH-ss-NH₂: ¹H NMR (400 MHz, CDCl₃) δ 4.98 (s, H, -C(O)NH-),

3.49 (q, J = 6.4 Hz, 2H, -C(O)NHCH₂-), 3.04 (t, J = 6.2 Hz, 2H, -CH₂NH₂), 2.89–2.70 (m,

4H, -CH₂ssCH₂-), 1.47 (s, 9H, t-Bu).

2.8. Synthesis of Boc-ss-PLA

The polymer is obtained via ring-opening polymerization of D, L- lactide with Boc-NH-ss-

NH₂as an initiator and SnCl₂as a catalyst [39]. Briefly, 0.5 g Boc-NH-ss-NH₂, 12 g D,L-

lactide, and 0.25 g of SnCl₂are added to a 100 mL single neck bottle and vacuumed for 6 h.

Then, the reaction was performed at 150 °C for 6 h. When the product is cooled to room-

temperature, 2 mL DCM is added to dissolve the product into a paste, and this followed by a

high amount of ice EtOH precipitation. The solid product is filtered and collected, and the

white product Boc-ss-PLA is obtained by vacuum drying (Boc-ss-PLA, 81% yield).

2.9. Synthesis of PEG-ss-PLA

First, Boc-ss-PLA (4 g) is dissolved in 100 mL DCM, and 2 mL trifluoroacetic acid (TFA)

is then added to the reaction while stirring. The reaction is subject to stirring at RM until the

Boc-ss-PLA completely reacts under monitoring with TLC. Subsequently, the solvent is

removed in vacuo, and the residue did not contain purified (PLA-ss-NH₂·TFA).

Second, COOH-PEG-COOH (2.2 g, 1 mmol) and DMAP（0.12 g, 1 mmol）are suspended in

anhydrous DCM (150 mL). DCC (0.74 g, 3.6 mmol) is dissolved in DCM (50 mL), and the

DCC solution is then added dropwise to the reaction. The mixture is stirred for 15 min, all the

unpurified PLA-ss-NH₂is then added, and the reaction mixture is stirred overnight at room

temperature for 24 h. Subsequently, 5 mL deionized water is added and stirred for 30 min to

quench the reaction. The reaction solution is filtered. The filtrate is concentrated under

vacuum, and the residue is purified by re-crystallization from diethyl ether (PEG-ss-PLA,

60% yield).2.10. Synthesis of TPP-PEG-ss-PLAand TPP-PEG-PLA

First, Boc-TPP (0.3 g, 0.53 mmol) is dissolved in 20 mL DMF, and 1 mL trifluoroacetic

acid (TFA) is added to the reaction while stirring. The reaction is stirred at RM until the Boc-

TPP reacts completely under monitoring with TLC. Subsequently, the solvent is removed in

vacuo, and the residue is did not contain purified (TPP-NH₂·TFA).

Subsequently, PEG-ss-PLA (2 g, 0.32 mmol) and DMAP (1 g, 8.37 mmol) are added into

DMF (50 mL). DCC (0.74 g, 3.60 mmol) is dissolved in DMF (20 mL) and added dropwise to

the reaction. The mixture is stirred for 15 min, all the unpurified TPP-NH₂.TFA is then added,

and the reaction mixture is stirred at room-temperature for 48 h. Furthermore, 5-mL deionized

water is added and stirred for 30 min to quench the reaction. The reaction solution is filtered.

The filtrate is concentrated under vacuum. The solvent is removed in vacuo, and the residue is

purified via re-crystallization from diethyl ether. (TPP-PEG-ss-PLA, 68% yield).

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In the same way, Boc-TPP (0.3 g, 0.53 mmol) is dissolved in 20 mL DMF, and 1 mL

trifluoroacetic acid (TFA) is added. The solvent is removed in vacuo, and the residue does not

contain purified (TPP-NH₂·TFA). PEG-PLA (2 g, 0.32 mmol) and DMAP (1 g, 8.37 mmol)

are added into DMF (50 mL). DCC (0.74 g, 3.60 mmol) is dissolved in DMF (20 mL) and

added dropwise to the reaction. The solvent is removed in vacuo, and the residue is purified

via re-crystallization from diethyl ether. (TPP-PEG-PLA, 75% yield).

2.11. Micelles Formation

The micelles were prepared according to previous report with some change [40]. Briefly, 5

mg of TPP-PEG-ss-PLA was dissolved in 5 mL of THF. The TPP-PEG-ss-PLA solution was

added dropwise into 10 mL of deionized water, the solution was allowed standing at 25℃ for

24 h under stirring. 1 mL of the CS water solution with 1 mg/mL was transferred into 10 mL

TPP-PEG-ss-PLA solutions (1mg/mL) and the CS/TPP-PEG-ss-PLA nanoparticles were

formed. DEX/TPP-PEG-ss-PLA nanoparticles were formed as same method. After the

treatment of dialysis, the obtained water solution was lyophilized to provide the nanoparticles

for the following experiments. The preparation process of CS/TPP-PEG-PLA@DOX, and

CS/TPP-PEG-ss-PLA@DOX were slightly different from above. Take CS/TPP-PEG-

PLA@DOX as example. First, DOX·HCl (1 mg) was dissolved in THF (10 mL), then a drop

of triethylamine was added to remove hydrochloric acid and DOX was obtained. After that,

the TPP-PEG-PLA (10 mg) was dissolved in the solution of DOX, the mixture was then

added dropwise to deionized water (10 mL) under high-speed stirring. After THF was

evaporated completely, the DOX-loaded micelles were transferred into dialysis bag (MWCO

1000) and dialyzed against deionized water to remove the unloaded DOX. After that, the

obtained water solution was lyophilized to obtain the micelle. The freeze-dried micelle sample

(TPP-PEG-PLA@DOX) was re-dissolved in water (1 mg mL^-1), 1 mL of the CS water

solution with 1 mg/mL was transferred into 10 mL TPP-PEG-ss-PLA micelle solutions (the

TPP-PEG-ss-PLA micelle solutions was obtained by resolving the freeze-dried micelle

sample (TPP-PEG-PLA@DOX) into water (1 mg mL^-1)) and the CS/TPP-PEG-PLA@DOX

nanoparticles were formed.

2.12. Characterizations

In the present study, ¹H Nuclear Magnetic Resonance (¹H NMR) spectra were measured via

the Bruker AM 300 apparatus. The solvents corresponded to CDCl₃and (CD₃)₂SO, and the

internal reference was Tetra-methylsilane (TMS). The average size and the Zeta potential of

all the micelles were measured by dynamic light scattering (DLS) (Zeta-Sizer, Malvern Nano-

ZS90, Malvern, U.K.). The morphology of micelles was detected by transmission electron

microscopy (TEM) that consisted of a JEOL 2010F instrument (JEOL Ltd., Japan).

Additionally, UV-vis spectrophotometer (UV-2550, Shimadzu, Japan) was used to detect the

amount of DOX that was loaded in the polymer. The critical micelle concentration (CMC)

was determined according to previous reports without changes by Fluoromax spectrometer (F-

7000, Hitach, Japan) using pyrene as a florescent probe [41]. Samples for the TEM were

prepared by drying a drop of the micellar solution (1 mg/mL) on a copper grid coated with

amorphous carbon and staining with a drop of phosphotungstic acid solution (2 wt%). Drug-

loading content (LC) and encapsulation efficiency (EE) of DOX-loaded micelles were

determined by a UV-vis spectrophotometer (UV-2550, Shimadzu, Japan). The freeze-dried

DOX-loaded micelle powders were re-dissolved in DMSO. The content of DOX in micelles

was determined by measuring its absorbance at 488 nm using a pre-established calibration

curve.

2.13. In vitro DOX Release

The in vitro DOX release was performed according to previous report with a slight change

[42]. 10 mg of TPP-PEG-ss-PLA@DOX or TPP-PEG-PLA@DOX micelle powders were

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dispersed in 10 mL PBS (pH 7.4) with 10 μM GSH, PBS (pH 7.4) with 10 mM GSH,

disodium hydrogen phosphate-citric acid buffer (pH 5.0) with 10 μM GSH, or disodium

hydrogen phosphate-citric acid buffer (pH 5.0) with 10 μM GSH in a dialysis bag (MWCO

1000). The dialysis bag was put into a tube which equipped with 30 mL corresponding

medium and the bag was immersed in the incubation media. 1 mL of solution was removed at

different time points (0, 3, 6, 9, 12, 24, 36, 48 and 72 h) for detected and replaced with 1 mL

of fresh medium. The cumulative amount of released DOX from the micelles was detected by

fluorescence spectrophotometer, and the percentages of released drug were plotted against

time.

2.14. CytocompatibilityAssay

The cytocompatibility of blank micelles was evaluated by the Alamar Blue (AB) assay and

live/dead staining which was performed according to previous report with slight change [3].

For the AB assay, 1 × 10⁴cells/well of HepG2 or HUVEC was seeded in 48-well plates. The

PEG-ss-PLA and CS/TPP-PEG-ss-PLA with different concentrations (25, 50, 100, 200 and

400 μg/mL) were added to each well for 24 h, respectively. For live/dead staining, 2 × 10⁴

cells/well of HepG2 or HUVEC was seeded in 24-well plates. The PEG-ss-PLA and CS/TPP-

PEG-ss-PLA micelles with different concentrations (25, 50, 100, 200 and 400 μg/mL) were

added to each well. The cytotoxicity study of free DOX, CS/TPP-ss-PLA nanoparticles and

DOX-loaded micelles with HepG2 cells were also carried out with similar method as

described above. The only difference was that the treated time was 48 h.

The cytotoxicity study of free DOX, CS/TPP-ss-PLA nanoparticles and DOX-loaded

micelles were also carried out with similar method as described above, the DOX dosages

varied from

0.075 to

μg/mL and the concentration of

CS/TPP-ss-PLA

nanoparticles varied from 15.6 to 1000 μg/mL. All groups were incubated with HepG2 for

48 h.

2.15. Flow Cytometry Studies

Flow cytometry was used to quantitatively evaluate the targeting effect of TPP. 1 × 10⁵

cells/well of HepG2 cells were seeded in 6-well plates and cultured at 37 ℃ for 24 h. The free

DOX and DOX-loaded micelles (DOX-equivalent dose: 5 μg/mL) were added and the cells

continued to incubate for 3 h at 37 ℃. The culture medium was applied as a blank control.

Cells were rinsed with PBS, harvested by trypsinization, centrifuged (1200 rpm, 4 min), and

re-suspended in PBS. The DOX fluorescence intensity was measured by a FACS Calibur flow

cytometer (BD Biosciences, U.S.A.).

2.16. In Vitro Cellular Uptake Studies

HepG2 cells were seeded in a confocal dish at a density of 5 × 10⁴cells/well and then

cultured with free DOX and different DOX-loaded micelles for 3 h after that, the medium in

the dishes was removed and the cells were washed with PBS for three times. Furthermore, the

cells were fixed with 2.5% glutaraldehyde for 30 min and washed with PBS twice. Then the

nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Finally, the cells were the

cells were observed by fluorescence microscopy (CKX41, Olympus, Japan).

2.17. The Mitochondrial Membrane Potential Analysis

Fluorescent probe JC-1 was employed to detect mitochondrial membrane potential changes

using confocal laser scanning microscopic (CLSM) and flow cytometry. For CLSM, 1 × 10⁵

cells/well of HepG2 cells were treated with PEG-ss-PLA and CS/TPP-PEG-ss-PLA micelles

in 6-well plates for 6h, then the plates were washed three times with PBS. After that, the cells

were incubated for 30 min with 1 µg/mL of JC-1 in culture medium at 37 °C in the dark. After

6 h incubation, the medium was removed and cells were washed with PBS for three times.

The cells were examined by CLSM (TCS SP5, Germany), JC-1 green was excited at 529 nm

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and the emission channel was set at 561 nm. Additionally, JC-1 red was excited at 490 nm,

and the emission channel was set at 633 nm.

For flow cytometry, 1 × 10⁵cells/well of HepG2 cells were treated for 6 h, 12 h with PEG-

ss-PLA and CS/TPP-PEG-ss-PLA micelles in 6-well plates, then the plates were washed three

times with PBS. After that, the cells were detached with trypsin-EDTA solution. Collected

cells were incubated for 30 min with 1 µg/mL of JC-1 in culture medium at 37 °C in the dark.

And then, the cells were washed with PBS and analyzed by flow cytometer (BD Biosciences,

U.S.A.).

2.18. The Measurement of Intracellular ROS Generation

The generation of intracellular ROS was measured via fluorescence microscopy using

DCFH-DA as the sensor. For mitochondrial membrane potential measurements, the HepG2

cells were seeded in a 6-well plate at a density of 1 × 10⁵cells/well. After incubated for 24 h,

PEG-ss-PLA and CS/TPP-PEG-ss-PLA micelles were added, then the cells were incubated for

another predetermined time. DCFH-DA was added (final concentration 1 × 10^-5M) and the

cells were incubated for 20 min. The cells were washed with PBS and then observed via

fluorescence microscopy, repeatedly.

2.19. Cellular Uptake, Intracellular Distribution and Co-LocationAnalysis

To evaluate whether a variety of stimuli in cells can enhance release of DOX, the DOX-

loaded DEX/TPP-PEG-ss-PLA and CS/TPP-PEG-ss-PLA micelles were incubated with

HepG2 cells. The intracellular distribution of the DOX were observed via confocal laser

scanning microscopic (CLSM). Briefly, HepG2 cells (1 ×10⁵cells per dish) were seeded in

petri dishes (35 mm × 10 mm, Corning Inc., New York), after incubated for 24 h, the free

DOX, CS/TPP-PEG-PLA@DOX and CS/TPP-PEG-ss-PLA@DOX micelles (DOX-

equivalent dose: 5 μg/mL) were added. After 3 h incubation, the medium was removed and

cells were washed with PBS for three times, then the mitochondria were stained with

Mitotracker Green (Beyotime Biotech, China). Images were obtained using a Leica

Microsystems CMS Gmbh (TCS SP5, Germany). DOX Emission of DOX moieties was

observed using a 488 nm laser with the emission channel was set to be 570 nm and expressed

as Red. Mitotracker Green was excited at 490 nm and the emission channel was set to be 516

nm. The amount of DOX in the cells and the Co-Location Analysis were finished by the

Image Pro-Plus 6.0 Image J software.

2.20. In vivo Detection of Mitochondrial Membrane Potential and Generation of Tumor

ROS

First, H22 (murine hepatic cancer cells) bearing Balb/c mice were i.v. injected with PEG-

ss-PLA or CS/TPP-PEG-ss-PLA micelles. After 24 h, JC-1 or DCFH-DA probes were injected

in situ. Approximately 0.5 h post-injection, the mice were anesthetized, and the tumor

epidermis was cut with a scalpel and imaged via a two-photon confocal microscope (A1RMP

Nikon). Subsequently, JC-1 green was excited at 529 nm and the emission channel was set at

561 nm. Additionally, JC-1 red was excited at 490 nm, and the emission channel was set at

633 nm; and DCFH-DA was excited at 529 nm and the emission channel was set at 561 nm.

The amount of JC-1 and DCFH-DA fluorescence in the tumor were measured via Image Pro-

Plus 6.0 software.

2.21. In vivo AntitumorActivity

H22 (murine hepatic cancer cells) bearing Balb/c mice were treated with saline, free DOX,

DOX-loaded micelles (CS/TPP-PEG-ss-PLA, PEG-ss-PLA@DOX, CS/TPP-PEG-

PLA@DOX and CS/TPP-PEG-ss-PLA@DOX) at a dosage of 5 mg DOX equiv./kg. A

solution of 0.9% NaCl and CS/TPP-PEG-ss-PLA blank micelles were used as controls. The

formulations were intravenously injected on days 0, 3 and 6. The tumor sizes were measured

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every three days, and the volume was calculated according to the formula: V = 0.5× a × b²,

where a is the length of the tumor and b is the width of the tumor. The mice were weighed,

and survival was recorded.

2.22. Histological Assessment

For the histological analysis (H&E and TUNEL staining), Balb/c mice were sacrificed at

day 21 after the first treatment, and the tumors were collected and fixed in 10% formalin and

embedded in paraffin blocks to prepare tumor sections at a thickness of 5 μm. After

deparaffinization, the tissue sections were stained with hematoxylin and eosin (H&E) and

terminal deoxynucleotidyltransferase mediated UTP end labeling (TUNEL) and were

visualized by an optical microscope (Olympus, Japan).

2.23. Statistical Analysis

SPSS software was used for the statistical data analysis. Data were expressed as means ±

SD. One way ANOVA was performed to determine statistical significance of the data. The

differences were considered significant for p values ** < 0.01, *** < 0.001.

3. Results and discussion

3.1. Characterization of Polymer

The intermediates and the resultant TPP functionalized polymer TPP-PEG-ss-PLA are

synthesized according to the routes shown in Scheme S1 in Supporting Information (SI).

First, the Boc-NH-ss-NH₂intermediate is achieved in a manner similar to a method in a

previous study [38], and the structure is confirmed by the H nuclear magnetic resonance (¹H

NMR) spectrum in Figure S1 in SI. Subsequently, Boc-ss-PLA is obtained by the ring-

opening polymerization of D, L-lactide using Boc-NH-ss-NH₂as an initiator, and its structure

and molecular weight (Mn = 5030 Da) are determined with H NMR (Figure S2 in SI).

Finally, a series of condensation reactions is used, and the resultant polymer TPP-PEG-ss-

PLA is obtained. The intermediates (Figure S3–S7) and TPP-PEG-ss-PLA with Mn = 7500

Da (Figure S8) are characterized by ¹H NMR to confirm the structures and compositions. As

shown in in Figure S8, we clearly observe peaks for the characteristic groups of the aromatic

ring group of the TPP (8.06–7.65 ppm), methylene group of the PEG (3.32 ppm), and methyl

group of the PLA component (1.45 ppm), thereby indicating that TPP-PEG-ss-PLA is

successfully synthesized.

3.2. Characterization of the Micelle

The micelle is obtained by the self-assembly of amphiphilic TPP-PEG-ss-PLA polymer. As

shown in the transmission electron microscopy (TEM) images and particle size distribution

determined by the dynamic laser scattering (DLS) method, it is clearly observed that the TPP-

PEG-ss-PLA blank micelles exhibit a well spherical shape, average size of 101.5 nm with a

nearly uniform distribution, and positive zeta potential of +20.8 mV (Figures 1A and 1B and

Table S1 in SI). To avoid elimination by the reticuloendothelial system (RES) in the blood

circulation, the CS are wrapped around the surface of micelles to reverse their positive charge

by electrostatic adsorption. Following the CS coating, the morphology of the CS/TPP-PEG-

ss-PLA micelles is still maintained as spherical, the size slightly increases to 135.0 nm; and

the zeta potential is fully reversed to -21.4 mV (Figure 1C and 1D). When the anticancer drug

DOX is encapsulated in the micelles, the shape, size, and zeta potential are almost unchanged.

The drug-loading content (LC) and encapsulation efficiency (EE) of all the micelles

approximately correspond to 6.0% and 70%, respectively (Table S1). The other micelles

including PEG-ss-PLA, TPP-PEG-PLA, and DOX loaded micelles are also prepared as

controls and their properties are also summarized in Table S1.

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3.3. pH and Redox Responsive Abilities

To demonstrate whether the CS/TPP-PEG-ss-PLA micelles drop off the CS layer via pH

responsiveness and regain mitochondrial targeting function after they enter tumor cells, we

measure the changes in both the particle size and zeta potential by varying pH values from 7.4

to 4.5. As shown in Figure 1G, the particle size and zeta potential do not significantly change

when the pH is adjusted from 7.4 to 6.5. When the pH is adjusted to 5.5, the particle size

reduces to ~100 nm; and the zeta potential reverses from -20 mV to +20 mV. To further verify

the pH sensitivity of CS/TPP-PEG-ss-PLA micelles, we investigate changes in the particle

size and potential with respect to time at pH 5.5. The results in Figure 1H indicate that the

surface charge of the CS/TPP-PEG-ss-PLA micelles reverse from negative to positive in less

than 5 minutes, and the micellar size changes similarly. This indicates that the shedding of CS

layer occurs immediately to expose TPP moieties in an acidic microenvironment. Additionally,

the introduction of disulfide bond as links between hydrophilic and hydrophobic segments in

TPP-PEG-ss-PLA polymer endows the micelles with redox-sensitivity in a high concentration

of glutathione (GSH) in the cytoplasm of tumor cells [41-43]. Therefore, the redox-

responsiveness of the micelles is also measured in a simulated cytoplasmic environment (pH

7.4 and GSH 10 mM). As shown in Figures 1E and 1F, a few large aggregates are formed,

and the size of TPP-PEG-ss-PLA micelles gradually changes from 101.5 nm to exceed 1000

nm in 24 h. The high redox-responsiveness accelerates the release of the encapsulated DOX

from the micelles. The in vitro DOX release profiles are shown in Figure 1I and indicate that

the cumulative release of DOX reaches ~84.1% in 72 h following the treatment of the PEG-

ss-PLA@DOX micelles by pH 7.4 and GSH 10 mM while the cumulative release of DOX

only reaches ~30% at pH 7.4 and GSH 10 μM. This is similar to a normal physiological

condition. The result observed that the micelles prevent the therapeutic agent from leaking in

blood circulation and also realize rapid drug release in tumor cells. Additionally, the

cumulative release of DOX from PEG-PLA@DOX micelles without disulfide bonds is

significantly lower in the simulated cytoplasmic microenvironment.

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Figure 1. Determination of the pH and reduction in the sensitivity of the micelles. Transmission electron

microscopy (TEM) images and changes in the size of corresponding nanoparticles: A-B) TPP-PEG-ss-PLA;

C-D) CS/TPP-PEG-ss-PLA; E-F) Morphology and size changes for CS/TPP-PEG-ss-PLA nanoparticles in

10 mM GSH at pH 7.4; G-H) Determination of pH sensitivity of the CS/TPP-PEG-ss-PLA nanoparticles

for different pH values and times (indicates ± standard deviation (SD), n = 3); I) In vitro DOX release

profiles (indicates ± SD, n = 3).

3.4. Cell Membrane Targeting

To verify whether the CS layer as a CD44 acceptor targets the cell membrane and enhances

the endocytosis of the nanoparticles, confocal laser scanning microscope (CLSM) is

employed to visualize the HepG2 cells following incubation with saline, free DOX, different

DOX-loaded nanoparticles, and CS coated DOX-loaded nanoparticles. Dextran (DEX) does

not actively target tumor cells overexpressed CD44 receptors and instead exhibits excellent

biocompatibility. It is selected as a control for coating TPP-PEG-ss-PLA@DOX micelles. As

shown in Figure S9, the cells are treated by DEX-coated nanoparticles and the red

fluorescence is extremely weak, thereby implying that only a low number of nanoparticles are

internalized due to the lack of cell membrane targeting. In the CS/TPP-PEG-ss-PLA@DOX

group, a significantly stronger red fluorescence is observed, thereby suggesting that additional

micelles are internalized via the receptor-mediated targeting effect. The flow cytometry

analysis shown in Figure S10 indicates a high degree of agreement. Additionally, we evaluate

the cytotoxicity of the blank micelles. The viability of HepG2 cells is investigated by Alamar

Blue (AB) assay and live/dead staining following incubation with the micelles with different

concentrations for 24 h. As shown in the fluorescence images in Figure S11 and AB results in

Figure S12, when the concentration of the micelles increases to 200 µg/mL, the cells grow in

a healthy manner, and a few dead cells are observed. The cell viability still exceeds 85%. The

cell viability slightly decreased to ~80% when the concentration of the CS/TPP-PEG-ss-PLA

micelles reaches 400 µg/mL, thereby indicating that the blank micelles exhibit inhibited effect

on tumor cell growth, and this is potentially because the TPP target the mitochondria and

induce the depolarization of the mitochondrial membrane.

3.5. Mitochondria-TargetingAbility

To investigate the mitochondria-targeting efficiency, we compare the behavior of

corresponding DOX-loaded nanoparticles with confocal laser scanning microscope (CLSM)

via co-localization analysis. The groups are adopted and include free DOX, CS/TPP-PEG-

PLA@DOX without redox-responsiveness, and CS/TPP-PEG-ss-PLA@DOX with redox-

responsiveness. In the CLSM images (Figure 2A), the mitochondria of HepG2 cells are

specially stained to green color with Mito traker green (MTG) dye, and DOX exhibits red

fluorescence. When DOX and mitochondria overlaps, yellow fluorescence appears due to the

merging of green and red colors. In the free DOX group, the green and red colors are

distinctively separated and located in the mitochondria and nucleus, respectively, thereby

suggesting that free DOX does not enter the mitochondria and instead diffuses into the

nucleus. The encapsulated DOX in the micelles are released rapidly because the micelles are

disassembled through the cleavage of the disulfide bonds in the micelle matrix under a high

GSH level in cytoplasm [44, 45]. The released DOX in cytoplasm diffuses into other

organelles. In the TPP functionalized micelle groups (CS/TPP-PEG-PLA@DOX, CS/TPP-

PEG-ss-PLA@DOX), yellow fluorescence is observed in the mitochondria in the merged

images, thereby indicating a good overlap between the green color for mitochondria and the

red color for DOX. Furthermore, only a low amount of red fluorescence is observed in the

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nucleus, thereby suggesting that only a low amount of DOX diffuses into the nucleus.

Additionally, the corresponding linear regions of interest intensity profiles across the white

line (marked in the CLSM images) are also employed to demonstrate the DOX distribution.

As shown in Figure 2B, the green and red peaks denote the mitochondria and DOX,

respectively. In a manner similar to the result in Figure 2A, only in TPP functionalized

micelle groups, green and red peaks completely overlap in the region of mitochondria, and a

few red peaks are obtained in the area of nucleus. The results also demonstrate that the CS

layer of the micelles is effectively removed, and the exposed TPP moieties in turn possess

excellent mitochondria- targeting ability, thereby leading to a specific accumulation of

nanocarriers at the mitochondrial region and lead to an improvement in the membrane

permeability of mitochondria and finally result in the direct diffusion of the released DOX

into mitochondria.

To quantitatively evaluate the DOX distribution in organelles, Pearson’s correlation (Rr)

and overlap coefficient (R) are utilized to demonstrate the correlation of two types of

fluorescence based on the fluorescence co-localization analysis. When the Rr value is closer

to 1, it indicates that the co-location of two types of fluorescence improves [46]. Additionally,

R is typically used to describe the degree of overlap between two types of fluorescence. The R

value ranges from 0 to 1 and increases in the R value increase the co-localization area [47]. As

shown in Figure 2C, the green and red colors also denote the fluorescence of mitochondria

and DOX, respectively. When compared to the free DOX group (Rr =0.67, R =0.68), the

CS/TPP-PEG-PLA@DOX group exhibits increased Rr (0.97) and R (0.97), and this indicates

that it can accurately target the mitochondria and release the DOX into the mitochondria. The

Rr and R values of CS/TPP-PEG-ss-PLA@DOX are 0.92 and 0.93, respectively, and this also

demonstrates the function. The Rr and R values of CS/TPP-PEG-ss-PLA@DOX are slightly

lower than those of CS/TPP-PEG-PLA@DOX. The reason is potentially because a part of

DOX is released in cytoplasm and diffused into nucleus due to the disassembly of

nanoparticles via the redox-responsiveness before the CS/TPP-PEG-ss-PLA@DOX targets

the mitochondria, which also means the DOX amount that can be delivered by the

nanoparticles into the mitochondria will be decreased. Simultaneously, the other small part of

the released DOX diffuses into nucleus, thereby leading to nuclear DNA damage. All the

aforementioned actions promote the apoptosis of tumor cells. Thus, all the results indicated

that the TPP functionalized micelles possess the capacity to target mitochondria and improve

membrane permeability, thereby leading to the direct diffusion of the released DOX into the

mitochondria.

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Figure 2. Mitochondria-targeting analysis. A) CLSM images for tracking DOX delivery in HepG2 cells

with MitoTracker-stained mitochondrion channel (green) or DOX channel (red). B, C) Co-localization

analysis.

3.6. Mitochondrial Membrane Potential Analysis

To determine whether the mitochondrial membrane potential decreases and leads to

improvements in the membrane permeability, a fluorescent probe, 5,5',6,6'-tetrachloro-

1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1), was employed. It is capable of

selectively entering mitochondria since it changes its color from red to green when the

membrane potential decreases [48, 48]. The normal mitochondria are accompanied by the red

fluorescence emission due to the formation of J-aggregates while the depolarized

mitochondria display green fluorescence due to the formation of J-monomers [50]. Thus,

changes in the membrane potential is measured by detecting the green/red fluorescence

intensity ratio. As shown in Figure 3A, the HepG2 cells treated with the micelles without TPP

targeting for 6 h still display red fluorescence, thereby suggesting that the mitochondrial

membrane potential is almost maintained as unchanged. Conversely, the cells treated with

CS/TPP-PEG-ss-PLA micelles gradually exhibit green fluorescence, and the red fluorescence

decreases or disappears, thereby implying decreases in the membrane potential. Flow

cytometry studies also confirmed that when the HepG2 cells are incubated with the CS/TPP-

PEG-ss-PLA micelles, the decrease in red fluorescence is evident and time-dependent

(Figures 3B–D). The ratio of the green to red fluorescence increase with increases in both the

incubation time and micelle concentration, and this also demonstrates that the TPP

functionalized micelle possesses the ability to decrease the mitochondrial membrane potential

(Figure S13). Thus, it increases the permeability of the mitochondrial membrane [51].

3.7. ROS Generation

When the permeability of mitochondrial membrane increases, the ROS is generated and

enters the cytoplasm [52]. Thus, we further examine whether the ROS is produced when the

TPP functionalized nanoparticles act on the mitochondria of cancer cells. The fluorescence

probe, 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA), is employed to measure the

generated ROS. The DCFH-DA is oxidized by ROS and in turn produces green fluorescence.

As shown in Figure 3E, after an incubation period of 6 h, a significantly stronger green

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fluorescence is clearly observed in the CS/TPP-PEG-ss-PLA group in contrast to the group

without TPP targeting, thereby indicating that several ROS are generated. Furthermore, as

shown in the quantitative result of the fluorescence intensity in Figure 3F, the intensity of the

mitochondria-targeted CS/TPP-PEG-ss-PLA is approximately thrice that without TPP target,

thereby suggesting that the cells increase ROS due to increases in the mitochondrial

membrane permeability. Additionally, with increases in the CS/TPP-PEG-ss-PLA

concentration, the green fluorescence intensity also increases (Figure 3G), thereby indicating

that the ROS generation in cytoplasm is concentration-dependent. The results also confirm the

improvement in mitochondrial membrane permeability. The IC₅₀value is the concentration

that results in 50% cell inhibition, and it is employed to evaluate the antitumor efficacy. As

shown in Figure 3H and Table S2, the blank CS/TPP-PEG-ss-PLA micelles display a IC₅₀

value similar to that of the DOX-loaded PEG-PLA micelles. Thus, the blank material exhibits

a certain amount of cytotoxicity to tumor cells. The result is ascribed to the fact that the

generated ROS results from TPP-induced mitochondrial membrane destruction and causes

cell apoptosis. Furthermore, the DOX-loaded CS/TPP-PEG-ss-PLA micelles exhibit an

extremely low IC₅₀value, thereby implying that a synergistic effect of the released DOX

exists and leads to mitochondrial DNA damage and generates ROS function on cell apoptosis.

At the same time, CS/TPP-PEG-PLA/DOX micelles exhibited a slightly higher IC50 than

CS/TPP-PEG-ss-PLA/DOX micelles, which was due to the cleavage of the disulfide bond,

and more DOX was released. It means the DOX amount that can be delivered by the micelles

into the mitochondria will be decreased. Simultaneously, the other small part of the released

DOX diffuses into nucleus, thereby leading to nuclear DNA damage. All the aforementioned

actions promote the apoptosis of tumor cells.

Figure 3. Invitro Analyses of the mitochondrial membrane potential and ROS generation. A) JC-1 staining

in HepG2 cells via treatment with different micelles for 6 h. B) Flow cytometric analyses of mitochondrial

membrane potential using JC-1 as indicator for HepG2 cells treated with various micelles at different

concentrations for 6 h and 12 h. Relative intensity of JC-1 red fluorescence for C) 6 h and D) 12 h

(indicates ± SD, n = 3). E) DCFH-DA staining in HepG2 cells after treatment with different micelles for 6

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h. F) Relative intensity of DCFH-DA green fluorescence. G) Mean fluorescence intensity (MFI) of the

DCFH-DA staining in HepG2 cells after treatment with CS/TPP-PEG-ss-PLA at different concentrations

(indicates ± SD, n = 3). H) Viability of HepG2 cells following incubation with free DOX and DOX-loaded

corresponding nanoparticles (indicates ± SD, n = 3).

Figure 4. In vivo analysis of mitochondrial membrane potential and ROS generation. Two-photon laser

confocal fluorescence microscopy images of A) in vivo JC-1 staining and B) DCFH-DA staining on Balb/c

mice bearing H22 tumor following treatment with different micelles for 24 h. C) Relative intensity of

DCFH-DA fluorescence from green.

3.8. In vivo Analysis of Mitochondrial Membrane Potential and ROS Generation

To verify whether the TPP modified nanoparticles decrease mitochondrial membrane

potentials and ROS generation in vivo, we intravenously injected the nanoparticles without

DOX on Balb/c mice bearing murine hepatic cancer (H22). At 24 h after injection, JC-1 or

DCFH-DA fluorescent probes are intravenously injected on the mice [53, 54]. As shown in

the two-photon laser confocal fluorescence microscopy images in Figure 4A, the tumor tissue

treated PEG-ss-PLA micelles without TPP targeting maintain the red fluorescence of JC-1

aggregates. However, the tumor treated with CS/TPP-PEG-ss-PLA micelles display the green

fluorescence of JC-1 monomers. The in vivo result is consistent with the in vitro data in

Figures 3A and 3B. It also observed that the TPP functionalized nanoparticles decrease the

mitochondrial membrane potential. Subsequently, we investigate the intracellular ROS

generation in tumor tissue by injection of DCFH-DA probe. As shown in Figure 4B,

significantly stronger green fluorescence in tumor treated with the CS/TPP-PEG-ss-PLA is

observed when compared with that without TPP targeting. Furthermore, the mean

fluorescence intensity indicates a significant difference between the CS/TPP-PEG-ss-PLA and

PEG-ss-PLA groups (Figure 4C), and this is in good agreement with the in vitro result in

Figures 3E–G. The result also suggests that the mitochondrial membrane potentials decrease

and the ROS is definitely generated in cytoplasm.

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3.9. In vivo Fluorescence Image

To further verify the cell membrane- targeting and redox-responsive capacities in vivo, we

inject the micelles on H22 tumor bearing nude mice, and monitor the fluorescence of DOX at

different time points. As shown in Figure 5A, in contrast to DEX/TPP-PEG-ss-PLA@DOX

group without active targeting, CS/TPP-PEG-ss-PLA@DOX group display stronger DOX

fluorescence in the tumor, thereby indicating the increased accumulation of the micelles.

Additionally, the DOX concentrations in the collected plasma and main organs including heart,

liver, spleen, lung, kidney, and tumor tissues at predesigned time points are measured via

fluorescence spectroscopy. The pharmacokinetic profiles of the DOX formulations are shown

in Figure 5B. As shown in the figure, in contrast to the free DOX, the elimination of DOX

from the blood is slower after it is encapsulated in micelles. Specifically, when the micelles

are coated by CS layer, the blood circulation time is significantly prolonged and increases to

~20 h. Biodistributions of DOX in main organs and tumor tissues versus time are shown in

Figure 5C–H. The DOX is typically accumulated in tumor, liver, heart, spleen, lung, and

kidneys at each time point. However, in contrast to the free DOX, other DOX nano-

formulations presented increased accumulation in tumor tissues. Furthermore, the

accumulation increases with increases in time. Specifically, the DOX accumulation of the CS

coated DOX-loaded micelles significantly increases and reaches ~7.5% at 24 h. This is ~1.7

times that of PEG-ss-PLA@DOX group without active targeting and ~9 times that of the free

DOX. All the results indicate that the CS coated micelles also exhibit excellent active

targeting ability.

Figure 5. In vivo and ex vivo biodistribution analyses. A) In vivo fluorescence images of H22 tumor

bearing nude mice following intravenous injection of various DOX formulations and ex vivo fluorescence

images of isolated tissues at 6 h post-injection. B) Pharmacokinetic profiles of total DOX, post-intravenous

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injection of various DOX formulations (indicates ± SD, n = 3). C-H) DOX distribution in the tumor, heart,

liver, spleen, lung, and kidneys (n = 3) (dose: 2 mg DOX/kg body weight, indicates ± SD, **P < 0.01).

3.10. In vivo Antitumor Efficacy

To establish the clinical translation potential of DOX-loaded nanoparticles, we perform

therapeutic studies via injecting nanoformulations on H22 tumor bearing Balb/c mice. The

antitumor efficacy is shown in Figure 6. As shown in Figures 6A and B, the mean tumor

volume continuously increases to ~1200 mm³, 700 mm³, 422 mm³, and 400 mm³following

21-d treatment with saline, PEG-ss-PLA@DOX, free DOX, and blank CS/TPP-PEG-ss-PLA,

respectively. However, the tumor growth is significantly inhibited via treatments of the DOX-

loaded TPP functionalized micelles. Specifically, the tumor volume in the CS/TPP-PEG-ss-

PLA@DOX group was only ~101 mm³, and this was only half that in CS/TPP-PEG-

PLA@DOX group. This result also indicates that the disulfide bonds can increase the release

of DOX, increasing the antitumor effect of DOX-loaded TPP functionalized micelles. It

should be noted that the blank CS/TPP-PEG-ss-PLA micelles represent comparative

antitumor efficacy, which is similar to that of the free DOX. This is also in accordance with

the in vitro antitumor effect with IC₅₀value of ~4 µg/mL DOX equivalent (Figure 3H). The

reason is mainly attributed to the fact that the TPP target mitochondria and the depolarization

of the mitochondrial membrane of tumor cells. The inhibition rate of tumor growth is

approximately 60% in CS/TPP-PEG-ss-PLA group and 90% in CS/TPP-PEG-ss-PLA@DOX

group (Figure 6D). The results indicate that the blank material CS/TPP-PEG-ss-PLA possess

a certain therapeutic effect by mitochondrial damage. Furthermore, the antitumor efficacy

further enhances significantly following the combination of DOX into the material. This

suggests the synergistic effect of the chemotherapy of DOX-inducing DNA damage and the

mitochondrial damage. The antitumor efficacy is further confirmed via a histological

evaluation of tumor tissues stained with hematoxylin and eosin (H&E) and terminal

deoxynucleotidyl transferase-mediated UTP end labeling (TUNEL). The apoptosis of tumor

cells is caused by the nanoparticles also can be clearly confirmed through TUNEL and H&E

analyses of tumor sections after treatment in vivo. Figure 6E shows extensive nuclear

shrinkage and fragmentation and massive tumor necrosis in the CS/TPP-PEG-ss-PLA@DOX

group, and this further indicates the optimal antitumor efficacy. Additionally, an evident

decrease in body weight is absent in all the groups with the exception of the free DOX group

(Figure 6C), thereby suggesting that the nanomedicines exhibit almost no systemic toxicity,

and the side effect of free DOX is overcome via the encapsulation of the materials.

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Figure 6. In vivo antitumor activity evaluation. A) Changes in the tumor volume and B) Monitored tumor

size of Balb/c mice bearing H22 tumor following intravenous saline injection, DOX·HCl, and

corresponding nanoparticles (2.0 mg DOX/kg body weight, indicates ± SD, n = 7), C) Body weight, D)

Tumor growth inhibition, E) TUNEL and H&E analyses of tumor sections after treatment. In TUNEL

staining, normal cells are stained blue, and apoptotic cells are stained brown. In H&E staining, nuclei are

stained blue, and the extracellular matrix and cytoplasm are stained red. (**P < 0.01 and ***P < 0.001)

4. Conclusions

In summary, we present a cell membrane/mitochondria dual targeting and pH/redox dual

responsive nanoparticle. The nanoparticle exhibits a long blood circulation time due to the

negative charged surface and enhanced specific internalization of tumor cells. In

lysosomes/endosomes, the negative charged CS layer falls off to expose the TPP in response

to the acidic microenvironment, and the nanoparticle targets and anchors into mitochondrial

outer membrane, thereby inducing the depolarization of mitochondrial membrane with

decreases in the ∆Ψ and increases in the mitochondria membrane permeability. Thus, the

overproduction of ROS in mitochondria promotes cell apoptosis, and the released DOX from

the disassembly of nanoparticles directly diffuses into mitochondria, thereby resulting in

mitochondrial DNA damage. All the aforementioned actions prompt the apoptosis of tumor

cells. Therefore, this work provides a new strategy for the design and engineering of a smart

nanoplatform for the mitochondrion therapy of malignant tumors.

Acknowledgements

This work was partially supported by the China National Funds for Distinguished Young

Scientists (51725303), the National Natural Science Foundation of China (21574105,

51603172, 81701831) and the Sichuan Province Youth Science and Technology Innovation

Team (Grant No.2016TD0026).

Appendix A. Supplementary data

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Supplementary data related to this article can be found online at http://xxxxxxxxxxxx.

Data availability statement

All data included in this study are available upon request by contact with the corresponding

author.

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Figure captions