Elsevier

Biomaterials

Volume 279, December 2021, 121189
Biomaterials

The safe and effective intraperitoneal chemotherapy with cathepsin B-specific doxorubicin prodrug nanoparticles in ovarian cancer with peritoneal carcinomatosis

https://doi.org/10.1016/j.biomaterials.2021.121189Get rights and content

Abstract

Intraperitoneal (IP) chemotherapy has shown promising efficacy in ovarian cancer with peritoneal carcinomatosis (PC), but in vivo rapid clearance and severe toxicity of free anticancer drugs hinder the effective treatment. Herein, we propose the safe and effective IP chemotherapy with cathepsin B-specific doxorubicin prodrug nanoparticles (PNPs) in ovarian cancer with PC. The PNPs are prepared by self-assembling cathepsin B-specific cleavable peptide (FRRG) and doxorubicin (DOX) conjugates, which are further formulated with pluronic F68. The PNPs exhibit stable spherical structure and cytotoxic DOX is specifically released from PNPs via sequential enzymatic degradation by cathepsin B and intracellular proteases. The PNPs induce cytotoxicity in cathepsin B-overexpressing ovarian (SKOV-3 and HeyA8) and colon (MC38 and CT26) cancer cells, but not in cathepsin B-deficient normal cells in cultured condition. With enhanced cancer-specificity and in vivo residence time, IP injected PNPs efficiently accumulate within PC through two targeting mechanisms of direct penetration (circulation independent) and systemic blood vessel-associated accumulation (circulation dependent). As a result, IP chemotherapy with PNPs efficiently inhibit tumor progression with minimal side effects in peritoneal human ovarian tumor-bearing xenograft (POX) and patient derived xenograft (PDX) models. These results demonstrate that PNPs effectively inhibit progression of ovarian cancer with peritoneal carcinomatosis with minimal local and systemic toxicities by high cancer-specificity and favorable in vivo PK/PD profiles enhancing PC accumulation.

Introduction

Peritoneal carcinomatosis (PC) is dissemination of cancerous tissues in peritoneal cavity, and common manifestation in digestive tract and gynecologic cancers including ovarian, gastric and colorectal cancers [1]. In particular, more than 70–80% of ovarian cancer patients present metastatic PC and it is one of the leading cause of cancer-related mortality with a median survial of 1–3 months [2]. Thus, the management of recurrent disease that has spread beyond the ovary to the peritoneal cavity is important challenge for ovarian cancer treatment [3]. Many clinical trials have indicated that intraperitoneal (IP) chemotherapy of anticancer drugs can be effective in ovarian cancer with PC [4]. The major advantage of IP chemotherapy can expose high concentrations of anticancer drugs to PC for a longer period of time, leading to effective tumor regression and favorable outcome [5,6]. Despite proven important clinical benefit of IP chemotherapy, it is not widely used due to high tissue absorption of anticancer drugs towards normal organs that cause both local and systemic toxicities [7,8]. Furthermore, rapid leakage of free anticancer drugs from peritoneal cavity reduce PC accumulation [9]. And, drug penetration is also limited to 3–5 mm from pheripheral region of tumors [10]. Thus, current IP chemotherapy should be applied in minimal residual diseases or forced patients to have cytoreductive surgery to reduce tumor burden to less than 3 mm prior to treatment for adequate anticancer efficacy [11].

Multiple nanoparticle-based IP chemotherapies have been developed to reduce non-specific toxicity and extend in vivo residence time of anticancer drugs for PC treatment [12]. Intraperitoneally administered nanoparticles containing anticancer drugs persist successfully in the peritoneal cavity and result in some systemic exposure through blood vessels, indicating high PC accumulation via two different targeting mechanisms of direct penetration (circulation independent) and systemic blood vessel-associated accumulation (circulation dependent) [13]. Thus, nanoparticle-based IP chemotherapy increased tumor exposure of anticancer drugs and improved anticancer efficacy compared to free anticancer drugs [9,14]. In addition, IP chemotherapy of anticancer drug loaded nanoparticles could be futher improved through pressurized intraperitoneal aerosol chemotherapy (PIPAC), that is known to ensure more homogeneous drug distribution with deeper drug penetration into peritoneal tumor nodules [15]. However, non-specific and rapid release of anticancer drugs from nanoparticles into peritoneal cavity and blood stream could induce not only local toxicity including abdominal pain, adhesion formation and chemical peritonitis, but also severe organ dysfunction attributed from systemic toxicity [16].

Herein, we propose cathepsin B-specific doxorubicin (DOX) prodrug nanoparticles (PNPs) for safe and effective IP chemotherapy in ovarian cancer with PC. To increase the cancer-specificity of IP chemotherapy, cathepsin B-specific DOX prodrug is prepared via direct conjugation of cathepsin B-specific cleavable peptide (Phe-Arg-Arg-Gly; FRRG) and DOX, resulting in FRRG-DOX. The amphiphilic FRRG-DOX molecules are self-assembled into nanoparticles through π−π stacking and hydrophobic interactions, and then FRRG-DOX prodrug nanoparticles (PNPs) are further formulated with pluronic F68 to enhance in vivo stability (Scheme 1a) [[17], [18], [19]]. Since the prodrug is non-toxic medication that can be converted to the active drug by enzymatic or chemical reaction at targeted cancer tissues, PNPs can prevent local and systemic toxicities in off-target sites caused by non-specific drug release through direct conjugation of peptide and drug [[20], [21], [22]]. In addition, among the various anticancer drugs, DOX is most suitable to make prodrugs conjugated with small molecule peptide since the conjugation with peptide dramatically inhibits the DNA intercalation of uncleaved-DOX in cells. We already reported that IV chemotherapy of PNPs efficiently eradicated tumors and reduced toxicity towards normal tissues compared with free DOX [23]. The PNPs were specifically cleaved and metabolized to free DOX in cathepsin B-overexpressing cancer cells, but they exhibited minimal cytotoxicity in cathepsin B-deficient normal cells. In addition, intravenously injected PNPs efficiently accumulated in targeted tumor tissues via enhanced permeability and retention (EPR) effect and exhibited potent therapeutic efficacy in tumor-bearing mice with minimum systemic toxicity in normal organs. In present study, we demonstrate that IP chemotherapy of PNPs can prolong in vivo residence time in peritoneal cavity with minimal in vivo toxicity towards normal organs. The extended residence time of PNPs in peritoneal cavity can effectively enhance accumulation within PC through two targeting mechanisms of direct penetration (circulation independent) and systemic blood vessel-associated accumulation (circulation dependent) of nanoparticles (Scheme 1b) [13]. Moreover, PNPs greatly reduce both local and systemic toxicities by selectively releasing free DOX only in cathepsin B-overexpressed PC cells compared to normal cells with innately low cathepsin B expression. In contrast, IP chemotherapy of free DOX result in only direct penetration to pheripheral region of tumor tissues and their rapid in vivo clearance reduce PC accumulation. Furthermore, high tissue absorption of free DOX towards normal tissues cause serious local and systemic toxicites. The effective anticancer efficacy of PNPs-based IP chemotherapy with minimal local and systemic toxicities is studied in peritoneal human ovarian tumor-bearing xenograft (POX) and patient derived xenograft (PDX) models. These results clearly suggest that PNPs provide an effective approach to overcome unavoidable disadvantages of current IP chemotherapy for PC treatment.

Section snippets

Materials

Doxorubicin (DOX) hydrochloride was purchased from FutureChem (Seoul, Republic of Korea). Pluronic F68, protease inhibitor cocktail, N,N-diisopropylethylamine (DIPEA), N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were purchased from Sigma-Aldrich (Oakville, Ontario, USA). N-terminal acylated Phe-Arg-Arg-Gly (Ac-FRRG) peptide was purchased from Peptron (Daejeon, Republic of Korea). Cathepsin B enzyme was purchased from R&D system (Minneapolis, MN, USA).

Animals

BALB/c nu/nu and BALB/c mice were purchased from NaraBio (Gyeonggi-do, Republic of Korea). Mice were bred under pathogen-free conditions at the Korea Institute of Science and Technology (KIST). All experiments with live animals were performed in compliance with the relevant laws and institutional guidelines of Institutional Animal Care and Use Committee (IACUC) in Korea Institute of Science and Technology (KIST), and IACUC approved the experiment (approved number of 2020–123). To generate a

Statistics

In this study, the statistical significance between two groups of experimental and control groups were analyzed using Student's t-test. One-way analysis of variance (ANOVA) was performed for comparisons of more than two groups, and multiple comparisons were analyzed using a Tukey-Kramer post-hoc test. Survival data was plotted via Kaplan-Meier curves and analyzed with the log-rank test. Statistical significance was marked with asterisk (*p < 0.05, **p < 0.01, ***< 0.001) in the figures.

Characterization of cathepsin B-specific doxorubicin prodrug nanoparticles (PNPs)

We designed the cathepsin B-specific doxorubicin prodrug nanoparticles (PNPs) to enhance the anticancer efficacy of IP chemotherapy without severe toxicity. First, the cathepsin B-specific prodrug was synthesized by conjugating cathepsin B-specific cleavable peptide (Phe-Arg-Arg-Gly; FRRG) to a doxorubicin (DOX) via 1 step EDC/NHS reation (Fig. S1). Successful synthesis of FRRG-DOX was confirmed via Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometer,

Conclusion

Herein, we proposed how overcoming the unavoidable limiations of current IP chemotherapy for treatment of ovarian cancer with peritoneal carcinomatosis with cathepsin B-specific doxorubicin prodrug nanoparticles (PNPs) via high cancer-specificity and enhanced tumor targeting ability with minimal toxicity. First, cathepsin B-specific doxorubicin prodrug (FRRG-DOX) was prepared by chemical conjugation of cathepsin B-specific cleavable peptide (Phe-Arg-Arg-Gly; FRRG) and doxorubicin (DOX), which

Author statement

Jinseong Kim: Validation, Formal analysis, Investigation, Data curation, Visualization. Man Kyu Shim: Methodology, Formal analysis, Data curation, Writing – original draft. Young-Jae Cho: Methodology, Investigation, Formal analysis, Data curation. Sangmin Jeon: Methodology. Yujeong Moon: Investigation. Jiwoong Choi: Investigation. Jaewan Lee: Investigation. Jeong-Won Lee: Conceptualization, Methodology, Visualization, Project administration, Funding acquisition, Supervision. Kwangmeyung Kim:

Data availability

All relevant data are available with the article and its Supplementary Information files, or available from the corresponding authors upon reasonable request.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported from the National Research Foundation (NRF) of South Korea, funded by the Ministry of Science (NRF-2019R1A2C3006283) of Republic of Korea, The Intramural Research Program of KIST, the KU-KIST Graduate School of Converging Science and Technology (Korea University & KIST) and the SMC-KIST Collaborative Research Program (SMC & KIST).

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