Elsevier

Journal of Controlled Release

Volume 220, Part B, 28 December 2015, Pages 704-714
Journal of Controlled Release

Efficacious delivery of protein drugs to prostate cancer cells by PSMA-targeted pH-responsive chimaeric polymersomes

https://doi.org/10.1016/j.jconrel.2015.08.058Get rights and content

Abstract

Protein drugs as one of the most potent biotherapeutics have a tremendous potential in cancer therapy. Their application is, nevertheless, restricted by absence of efficacious, biocompatible, and cancer-targeting nanosystems. In this paper, we report that 2-[3-[5-amino-1-carboxypentyl]-ureido]-pentanedioic acid (Acupa)-decorated pH-responsive chimaeric polymersomes (Acupa-CPs) efficiently deliver therapeutic proteins into prostate cancer cells. Acupa-CPs had a unimodal distribution with average sizes ranging from 157–175 nm depending on amounts of Acupa. They displayed highly efficient loading of both model proteins, bovine serum albumin (BSA) and cytochrome C (CC), affording high protein loading contents of 9.1–24.5 wt.%. The in vitro release results showed that protein release was markedly accelerated at mildly acidic pH due to the hydrolysis of acetal bonds in the vesicular membrane. CLSM and MTT studies demonstrated that CC-loaded Acupa10-CPs mediated efficient delivery of protein drugs into PSMA positive LNCaP cells leading to pronounced antitumor effect, in contrast to their non-targeting counterparts and free CC. Remarkably, granzyme B (GrB)-loaded Acupa10-CPs caused effective apoptosis of LNCaP cells with a low half-maximal inhibitory concentration (IC50) of 1.6 nM. Flow cytometry and CLSM studies using MitoCapture™ revealed obvious depletion of mitochondria membrane potential in LNCaP cells treated with GrB-loaded Acupa10-CPs. The preliminary in vivo experiments showed that Acupa-CPs had a long circulation time with an elimination phase half-life of 3.3 h in nude mice. PSMA-targeted, pH-responsive, and chimaeric polymersomes have appeared as efficient protein nanocarriers for targeted prostate cancer therapy.

Graphical abstract

Long-circulating and PSMA-targeting pH-sensitive degradable chimaeric polymersomes (Acupa-CPs) efficiently load, deliver and release potent apoptotic proteins like granzyme B to PSMA-overexpressing prostate cancer cells, inducing specific and superior anticancer effects.

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Introduction

Protein drugs are one of the most potent biotherapeutics that have shown a tremendous potential in cancer therapy [1], [2], [3]. In compared with chemotherapeutics, protein drugs have advantages of high therapeutic activity, high selectivity, and low toxicity to healthy cells. In the past years, many apoptotic proteins such as TRAIL, herceptin, cytochrome C (CC) and granzyme B (GrB) have been investigated for cancer therapy [4], [5], [6], [7]. The direct administration of protein drugs, however, resulted in typically diminished therapeutic efficacy due to rapid degradation and elimination, poor bioavailability, possible immune response, and low cell permeability. In order to potentiate protein therapy for cancers, different nanosystems ranging from liposomes, nanocapsules, lipid-like nanoparticles, to nanogels have been designed and investigated in vitro and in vivo for intracellular delivery of apoptotic proteins such as antibodies, CC, apoptin, caspase 3, ricin and saporin [8], [9], [10], [11], [12]. Ying et al. recently reported that micellar herceptin nanocomplexes demonstrated longer blood half-life, better tumor selectivity and growth reduction than free herceptin [13]. In particular, polymersomes containing a watery interior are among the most ideal nanocarriers for loading and delivery of therapeutic proteins [14], [15]. It should be noted, nevertheless, that current polymersomes are not optimal as they usually show low protein loading, poor cellular uptake, and/or slow protein release in the target cells.

Enhanced drug and protein release can be achieved via designing stimuli-sensitive polymersomes [16], [17], [18], [19]. In the past years, several stimuli-sensitive polymersome systems that release proteins in response to a signal like acidic pH, glucose or cytoplasmic glutathione have been developed [20], [21], [22], [23]. pH-Sensitive polymersomes are especially appealing because there exists a lower pH in the endosomes and lysosomes of cancer cells (pH 4.0–6.0) as well as in the tumor tissues (pH 6.8–7.2) [24], [25]. We reported previously that acid-degradable polymersomes based on poly(ethylene glycol)-b-poly(2,4,6-trimethoxybenzylidene-pentaerythritol carbonate) (PEG-PTMBPEC) diblock copolymer exhibited accelerated release of chemotherapeutics like doxorubicin hydrochloride (hydrophilic) and paclitaxel (hydrophobic) [26]. However, vesicles have a typically low loading efficacy toward hydrophilic drugs including proteins due to a small volume partition of vesicular core. We found that chimaeric polymersomes (CPs) self-assembled from asymmetric PEG-PCL-PDEA triblock copolymers could efficiently encapsulate various proteins due to active interactions between proteins and PDEA inside of the vesicles [27].

In order to achieve targeted cancer treatment with therapeutic proteins, polymersomes have to be functionalized with cancer homing ligands [28]. In the past years, polymersomes decorated with various ligands like antibody, peptide, or folate have been explored for tumor-targeted chemotherapeutics or siRNA delivery [29], [30], [31], [32], [33]. It should be noted, however, that there are few reports on tumor-targeted polymersomes for controlled delivery of therapeutic proteins. Kokkoli et al. reported that PR_b peptide coupled polymersomes loaded with tumor necrosis factor-alpha showed dramatically enhanced cytotoxicity to LNCaP prostate cancer cells as compared to non-targeting polymersomes [15]. Galactose-decorated reduction-sensitive polymersomes were found to efficiently load and chaperone GrB into hepatocellular carcinoma cells [34].

In this article, we report that 2-[3-[5-amino-1-carboxypentyl]-ureido] pentanedioic acid (Acupa)-decorated pH-responsive chimaeric polymersomes (Acupa-CPs) efficiently deliver therapeutic proteins, CC and GrB, into prostate cancer cells (Scheme 1). Prostate cancer is one of the most common non-cutaneous malignancies in men and is fatal following metastasis. The prostate specific membrane antigen (PSMA) is a clinically validated transmembrane receptor that is over-expressed on the surface of prostate cancer cells as well as in the neovasculature of nearly all non prostate solid tumors [35], [36]. Acupa-installed nanoparticles have proved to actively target to prostate cancer in vitro and in vivo [36], [37], [38]. Notably, Acupa-directed docetaxel-loaded PEG-PLGA nanoparticles have entered Phase II clinical trials for advanced tumor treatments. Here, Acupa-CPs were constructed from poly(ethylene glycol)-b-poly(2,4,6-trimethoxybenzylidene-pentaerythritol carbonate)-b-poly(succinic acid carbonate) (PEG-PTMBPEC-PSAC) and Acupa-PEG-PTMBPEC-PSAC triblock copolymers. The preparation of pH-sensitive degradable Acupa-CPs, loading and pH-triggered release of proteins, PSMA-targeted intracellular protein release, in vitro antitumor activity of protein-loaded Acupa-CPs, as well as in vivo circulation of Acupa-CPs were investigated.

Section snippets

Synthesis of Acupa-PEG-PTMBPEC-PSAC

Acupa-PEG-PTMBPEC-PSAC was synthesized in three steps. Firstly, under a nitrogen atmosphere, to a stirred anhydrous DCM (7.0 mL) solution of NHS-PEG-OH (6.6 kg/mol, 0.33 g, 50 μmol) and TMBPEC (0.75 g, 2.2 mmol) was added a stock solution of zinc bis[bis(trimethylsilyl)amide] in DCM (20 mg/mL, 0.75 mL, 39 μmol). The reaction vessel was sealed and placed in an oil bath thermostated at 40 °C. The polymerization proceeded with magnetic stirring for 4 d. A sample was taken for the determination of monomer

Results and discussion

The clinical translation of polymersomal protein drugs to cancer therapy demands high protein loading, selective recognition and internalization by cancer cells, and fast release of protein inside the cancer cells. It is also equally important that polymersomes are based on biocompatible and degradable polymers and are easy to prepare. To this end, we have designed and developed Acupa-decorated pH-sensitive degradable chimaeric polymersomes (Acupa-CPs) for PSMA-targeted delivery of apoptotic

Conclusions

We have demonstrated that PSMA-targeting pH-sensitive degradable chimaeric polymersomes (Acupa-CPs) could efficiently load, deliver and release apoptotic proteins including cytochrome C and granzyme B to PSMA-overexpressing LNCaP cells, resulting in specific and exceptional anticancer effects. The initial in vivo studies showed that Acupa-CPs had a long circulation time. These multifunctional polymersomes offer several appealing features for targeted protein therapy: (i) they are readily

Acknowledgments

This work is financially supported by research grants from the National Natural Science Foundation of China (NSFC 51273139 and 51473111), the National Science Fund for Distinguished Young Scholars (NSFC 51225302), Ph.D. Programs Foundation of Ministry of Education of China (20133201110005), the Major Program of the Natural Science Foundation of Jiangsu Province (14KJA150008) and a Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

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