*Article* **Mitochondria-Targeted Delivery of Camptothecin Based on HPMA Copolymer for Metastasis Suppression**

**Xiaoli Yi † , Yue Yan † , Xinran Shen, Lian Li and Yuan Huang \***

> Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, No. 17, Block 3, South Renmin Road, Chengdu 610041, China; yixiaoli728@126.com (X.Y.); xinwen93696@126.com (Y.Y.); yy18224019649@163.com (X.S.); liliantripple@163.com (L.L.)

**\*** Correspondence: huangyuan0@163.com

† These authors contributed equally to this work.

**Abstract:** Poor anti-metastasis effects and side-effects remain a challenge for the clinical application of camptothecin (CPT). Mitochondria can be a promising target for the treatment of metastatic tumors due to their vital roles in providing energy supply, upregulating pro-metastatic factors, and controlling cell-death signaling. Thus, selectively delivering CPT to mitochondria appears to be a feasible way of improving the anti-metastasis effect and reducing adverse effects. Here, we established a 2-(dimethylamino) ethyl methacrylate (DEA)-modified *N*-(2-hydroxypropyl) methacrylamide (HPMA) copolymer–CPT conjugate (P-DEA-CPT) to mediate the mitochondrial accumulation of CPT. The mitochondria-targeted P-DEA-CPT could overcome multiple barriers by quickly internalizing into 4T1 cells, then escaping from lysosome, and sufficiently accumulating in mitochondria. Subsequently, P-DEA-CPT greatly damaged mitochondrial function, leading to the reactive oxide species (ROS) elevation, energy depletion, apoptosis amplification, and tumor metastasis suppression. Consequently, P-DEA-CPT successfully inhibited both primary tumor growth and distant metastasis in vivo. Furthermore, our studies revealed that the mechanism underlying the anti-metastasis capacity of P-DEA-CPT was partially via downregulation of various pro-metastatic proteins, such as hypoxia induction factor-1α (HIF-1α), matrix metalloproteinases-2 (MMP-2), and vascular endothelial growth factor (VEGF). This study provided the proof of concept that escorting CPT to mitochondria via a mitochondrial targeting strategy could be a promising approach for anti-metastasis treatment.

**Keywords:** camptothecin; metastasis; mitochondria; 2-(dimethylamino) ethyl methacrylate

## **1. Introduction**

CPT is a promising chemotherapeutic agent due to its potent inhibitory effect against DNA topoisomerase [1]. Although targeted delivery of CPT has been reported to improve its cytotoxicity, the anti-metastasis effect is poor [2–4]. Furthermore, recent studies disclosed that nuclear DNA damage by CPT could induce a massive release of double-stranded DNA (dsDNA), which could subsequently stimulate a strong immune response to initiate the intestinal diarrhea, a life-threatening side-effect of camptothecin [5,6]. Thus, exploring new strategies to improve its anti-metastasis efficacy while reducing its side-effects is needed.

Due to the imperative roles in regulating cellular metabolism and cell-death signaling [7,8], mitochondria not only provide various metabolites and antiapoptotic proteins for rapid tumor growth but also facilitate the migration and invasion of tumor cells by offering abundant energy and upregulating pro-metastatic factors [9–11]. Increasing evidence has shown that targeted induction of mitochondria dysfunction by therapeutics (i.e., doxorubicin, lonidamine, and metformin) hold tremendous potential for suppressing both primary tumors and metastases [12–14]. Notably, CPT can be a cellular respiration inhibitor to impair mitochondrial without causing side-effects such as diarrhea [15,16].

**Citation:** Yi, X.; Yan, Y.; Shen, X.; Li, L.; Huang, Y. Mitochondria-Targeted Delivery of Camptothecin Based on HPMA Copolymer for Metastasis Suppression. *Pharmaceutics* **2022**, *14*, 1534. https://doi.org/10.3390/ pharmaceutics14081534

Academic Editors: Emanuela Fabiola Craparo and Marta Gonzá-lez-Álvarez

Received: 16 May 2022 Accepted: 20 July 2022 Published: 23 July 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Thus, selectively delivering CPT to mitochondria appears to be a feasible way of improving the anti-metastasis effect and reducing adverse effects. ing the anti-metastasis effect and reducing adverse effects. Free drugs rely on simple diffusion to randomly interact with the organelles of tumor cells [17]. Thus, the amount of CPT that accumulates in mitochondria is very limited due

(i.e., doxorubicin, lonidamine, and metformin) hold tremendous potential for suppressing both primary tumors and metastases [12–14]. Notably, CPT can be a cellular respiration inhibitor to impair mitochondrial without causing side-effects such as diarrhea [15,16]. Thus, selectively delivering CPT to mitochondria appears to be a feasible way of improv-

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 2 of 16

Free drugs rely on simple diffusion to randomly interact with the organelles of tumor cells [17]. Thus, the amount of CPT that accumulates in mitochondria is very limited due to the multiple intracellular barriers to reaching mitochondria and the extremely poor permeability of the mitochondrial inner membrane, which represent formidable hurdles [18,19]. Recently, various ligands have been reported to improve mitochondrial accumulation, such as triphenylphosphonium bromide, mitochondrial penetration peptide, and mitochondrial targeting sequence [20–22]. However, those lipophilic moieties might deteriorate the solubility of the highly hydrophobic CPT. 2-(Dimethylamino) ethyl methacrylate, a hydrophilic small molecular containing a tertiary amino group, might be a promising ligand to facilitate the transport of CPT to mitochondria [23]. Nevertheless, the modification content of DEA for effective mitochondria targeting remains unknown. to the multiple intracellular barriers to reaching mitochondria and the extremely poor permeability of the mitochondrial inner membrane, which represent formidable hurdles [18,19]. Recently, various ligands have been reported to improve mitochondrial accumulation, such as triphenylphosphonium bromide, mitochondrial penetration peptide, and mitochondrial targeting sequence [20–22]. However, those lipophilic moieties might deteriorate the solubility of the highly hydrophobic CPT. 2-(Dimethylamino) ethyl methacrylate, a hydrophilic small molecular containing a tertiary amino group, might be a promising ligand to facilitate the transport of CPT to mitochondria [23]. Nevertheless, the modification content of DEA for effective mitochondria targeting remains unknown. Herein, we designed a DEA-modified HPMA copolymer–CPT conjugate (P-DEA-

Herein, we designed a DEA-modified HPMA copolymer–CPT conjugate (P-DEA-CPT) consisting of (1) HPMA copolymer acting as a carrier and (2) DEA acting as the mitochondria-targeting moiety (Scheme 1). DEA was decorated on the side-chain, and its modification degree was screened to ensure sufficient mitochondria accumulation. P-DEA-CPT greatly enhanced mitochondrial location of CPT in cancer cells. Subsequently, P-DEA-CPT induced mitochondrial dysfunction, thereby efficiently curbing the growth and metastasis of breast cancer. Thus, our study unleashed the potential for anti-metastasis treatment of CPT via a mitochondria-targeted delivery system. CPT) consisting of (1) HPMA copolymer acting as a carrier and (2) DEA acting as the mitochondria-targeting moiety (Scheme 1). DEA was decorated on the side-chain, and its modification degree was screened to ensure sufficient mitochondria accumulation. P-DEA-CPT greatly enhanced mitochondrial location of CPT in cancer cells. Subsequently, P-DEA-CPT induced mitochondrial dysfunction, thereby efficiently curbing the growth and metastasis of breast cancer. Thus, our study unleashed the potential for anti-metastasis treatment of CPT via a mitochondria-targeted delivery system.

**Scheme 1.** Schematic illustration of P-DEA-CPT for damaging mitochondria and suppressing metastasis. After internalized into cancer cells, P-DEA-CPT ① escaped from lysosome, ② targeted mitochondria, ③ damaged mitochondria and ④ finally induced cell apoptosis. **Scheme 1.** Schematic illustration of P-DEA-CPT for damaging mitochondria and suppressing metastasis. After internalized into cancer cells, P-DEA-CPT <sup>1</sup> escaped from lysosome, <sup>2</sup> targeted mitochondria, <sup>3</sup> damaged mitochondria and <sup>4</sup> finally induced cell apoptosis.

#### **2. Materials and Methods**

#### *2.1. Materials*

Camptothecin (CPT) (>98%) and 2-(dimethylamino) ethyl methacrylate (DEA) were acquired from Giant Medical Technology Co., Ltd. (Chengdu, China), whereas *N*-(3 aminopropyl) methacrylamide hydrochloride was purchased from Bide Pharmaceutical Technology Co., Ltd. (Shanghai, China). Lyso-tracker Red was provided by Thermo Fisher Scientific (Shanghai, China). MitoTracker Red, Mitochondrial Membrane Potential Assay Kit, Reactive Oxygen Species Assay Kit, ATP Assay Kit, Caspase 3 and Caspase 9 Activity Assay Kit, and Tissue Mitochondria Isolation Kit were all obtained from Beyotime Biotechnology Co., Ltd. (Shanghai, China). All other reagents were of analytical grade or above.

#### *2.2. Cells and Animals*

Murine breast cancer cells (4T1) and human umbilical vein endothelial cells (HUVECs) were obtained from Icell Biotech Co., Ltd. (Shanghai, China) and the Chinese Academy of Science Cell Bank for Type Culture Collection (Shanghai, China), respectively. Cells were incubated in a homothermal cell incubator (37 ◦C) with 5% CO2. RPMI 1640 medium (Gibco, Invitrogen Co., Grand Island, NY, USA) supplemented with 10% (*v*/*v*) fetal bovine serum (FBS) and 1% (*v*/*v*) antibiotics (penicillin–streptomycin) was used for 4T1 cells, and Dulbecco's modified Eagle's medium (Gibco, Invitrogen) supplemented with 10% (*v*/*v*) fetal bovine serum (FBS) and 1% (*v*/*v*) antibiotics (penicillin–streptomycin) was used for HUVECs.

Female BALB/c mice (6 to 10 weeks) were provided by Chengdu Dashuo Experimental Animal Co., Ltd. (Chengdu, China). All animal experiments strictly abided by the Guidelines of Medical Ethics Committee of Sichuan University.

## *2.3. Synthesis, Characterization, and Mitochondria-Targeting Capacity of HPMA Copolymers with Different Modification Ratios of DEA*

FITC-labeled HPMA copolymers conjugated with different 2-(dimethylamino) ethyl methacrylate (DEA) ratios were synthesized, and their mitochondrial accumulation was evaluated. Firstly, *N*-(2-hydroxypropyl) methacrylamide (HPMA) and *N*-(3-aminopropyl) methacrylamide–fluorescein isothiocyanate monomer (APMA–FITC) were synthesized in the same way as our previous study [24]. Then, FITC-labeled HPMA polymers with various DEA modification amounts were obtained by direct radical polymerization of monomers. Briefly, the monomers (APMA–FITC:DEA:HPMA = 5:0~13:95~82 mol.%) and azobisisobutyronitrile (2 wt.%) as the initiator were dissolved in dimethyl sulfoxide and stirred for 24 h at 50 ◦C under argon atmosphere. Products were precipitated into diethyl ether and freeze-dried after being purified by dialysis. The obtained HPMA conjugates modified with 0%, 5%, 10% and 13% molar ratios of DEA were defined as PFITC, P-DEA (5%)-FITC, P-DEA (10%)-FITC, and P-DEA (13%)-FITC, respectively. Next, a Fast Protein Liquid Chromatograph (FPLC, GE Healthcare Life Science, Piscataway, NJ, USA) was used to detect the molecular weight and polydispersity of these copolymers. The zeta potential of the copolymers was estimated by Zetasizer Nano ZS90 at 25 ◦C (Malvern Instruments, Malvern, UK). The amount of FITC contained in these copolymers was determined via ultraviolet spectroscopy GENESYS 180 (Thermo fisher technologies, South San Francisco, CA, USA).

Then, mitochondria-targeting capacity of various HPMA copolymers was evaluated in 4T1 cells. Briefly, after being incubated with the above-obtained copolymers (FITC dose, <sup>10</sup> <sup>µ</sup>g·mL−<sup>1</sup> ) for 4 h, the mitochondria of 4T1 cells were extracted by grinding the cells in mitochondria extraction reagent under ice bath 20 times, and cell debris was removed by centrifuging at 600× *g* for 10 min at 4 ◦C. Next, the obtained supernatant containing mitochondria was centrifuged at 11,000× *g* for 15 min at 4 ◦C, and the mitochondria pellets were collected. Finally, the fluorescence intensity of FITC in mitochondria was quantitatively determined via flow cytometry (FACS Calibur, BD, Franklin Lakes, NJ, USA).

Furthermore, the safety of these copolymers was investigated in both 4T1 tumor cells and HUVECs. After seeding in 96-well plates, 4T1 cells and HUVEC were treated with P-FITC, P-DEA (5%)-FITC, P-DEA (10%)-FITC, and P-DEA (13%)-FITC at various predetermined concentrations for 48 h. Then, fresh MTT agent was added and cultured for another 4 h. Finally, the amount of formazan in each well was determined by Varioskan Flash 902-ULTS (Thermo Scientific, Sunnyvale, CA, USA) after being dissolved in 200 µL of dimethyl sulfoxide (DMSO). The relative cell viability was calculated as the absorption value of experimental wells reverse that in the drug-free medium treated group.

## *2.4. Synthesis and Characterization of HPMA Copolymer-CPT Conjugates*

The azelaic acid–camptothecin conjugate (LA–CPT) and *N*-(3-aminopropyl) methacrylamide hydrochloride–azelaic acid–camptothecin (APMA–LA–CPT) monomer were synthesized as described in our previous study [23]. Subsequently, CPT-loaded HPMA copolymers with (13% molar ratio, P-DEA-CPT) or without DEA modification (P-CPT) were synthesized according to the same procedure mentioned above. The molecular weight, polydispersity, zeta potential, and CPT loading capacity of P-CPT and P-DEA-CPT were evaluated using the same method in Section 2.3.

#### *2.5. Cellular Uptake, Lysosome Escape, and Mitochondrial Targeting of HPMA Copolymer–CPT Conjugates*

The 4T1 cells were treated with free CPT, P-CPT and P-DEA-CPT (equivalent CPT dose, 20 <sup>µ</sup>g·mL−<sup>1</sup> ) for 4 h. Then, 4T1 cells were harvested, and the fluorescence intensity of CPT was qualitatively observed via a laser scanning confocal microscope (CLSM, Zeiss LSM 800, Oberkochen, Germany) and quantitatively detected via flow cytometry.

Then, whether HPMA copolymer–camptothecin conjugates could escape from lysosome and accumulate in mitochondria was investigated. The 4T1 cells were incubated with P-CPT or P-DEA-CPT (equivalent CPT dose, 20 <sup>µ</sup>g·mL−<sup>1</sup> ) for 4 h. After being labeled with Mito-Tracker Red or Lyso-Tracker Red, cells were visualized under CLSM.
