Elsevier

Biomaterials

Volume 145, November 2017, Pages 223-232
Biomaterials

Polyglycerolated nanocarriers with increased ligand multivalency for enhanced in vivo therapeutic efficacy of paclitaxel

https://doi.org/10.1016/j.biomaterials.2017.08.042Get rights and content

Abstract

Despite the excellent biocompatibility and antifouling effect of poly(ethylene glycol) (PEG), the high steric hindrance, limited chemical functionality, and low ligand multivalency of PEGylated nanocarriers often lead to inefficient cell targeting and intracellular trafficking. Hence, a new structure of hydrophilic corona allowing a higher ligand density without loss of excellent biocompatibility is highly desirable. Here we introduce tumor-targeted polyglycerolated (PGylated) nanocarriers that dramatically enhance the in vivo therapeutic efficacy of incorporated paclitaxel simply by increasing the surface density of hydrophobic tumor-targeting ligands. Linear polyglycerol-poly (ε-caprolactone) block copolymer (PG-b-PCL) is used to prepare PGylated lipiodol nanoemulsions, where PG serves as a corona conjugated with a large number of folic acid (FA) for efficient tumor targeting. Unlike FA-PEGylated nanoemulsions, FA-PGylated nanoemulsions can display a larger number of FA without structural destabilization. This property enables excellent anti-cancer activities and effective tumor regression in a cervical cancer xenograft murine model at a cumulative drug dose of ∼5 mg kg−1, which is about four fold smaller than that of commercial Taxol formulation. This study highlights the importance of surface chemistry of nanocarriers that enable multivalent ligand functionalization and high tolerance to the conjugation of hydrophobic ligands, which make PG as a very effective hydrophilic corona for in vivo drug delivery.

Introduction

For the past decades, various polymeric micelles and nanoparticles prepared via the self-assembly of amphiphilic block copolymers have been extensively studied for efficient in vivo drug delivery [1]. In particular, polyethylene glycol (PEG)-modified, or PEGylated, biodegradable polymers, including poly (ε-caprolactone) (PCL) [2], [3], polylactide [4], poly(lactide-co-glycolide) [5], [6], and human serum albumin [7], [8] have been developed to fabricate long-circulating colloidal nanocarriers. It is well established that PEG efficiently prevents the non-specific adsorption of serum proteins and aggregation of nanoparticles in the blood stream via steric hindrance, effectively increasing their blood circulation time and cell-targeting efficiency [9], [10]. However, recent studies revealed that a long PEG chain can hinder the cellular uptake of nanoparticles and endosomal escape partially due to its neutrality and lack of responsiveness to biological stimuli [11], [12]. Moreover, PEG has a very low physical tolerance to the conjugation of hydrophobic ligands because highly flexible, neutral PEG chains allow the ligands to be aggregated through hydrophobic interactions on the surface of nanocarriers, rapidly reducing their dispersion stability. Ironically, the ligand multivalency is particularly required for PEGylated nanoparticles to achieve efficient cellular uptake and tumor targeting selectivity, which can be adversely affected by the excellent antifouling and anti-adhesion properties of PEG. Thus, it is highly desirable to explore new hydrophilic corona polymers that can overcome the limitations of PEG for drug delivery applications.

In this regard, hyperbranched polyglycerol (PG) has received increasing attention as an alternative hydrophilic polymer to PEG for biomedical and pharmaceutical applications due to its unique chemical properties and molecular structure [13], [14], [15], [16]. These include the ability to form dendritic architecture, a large number of hydroxyl groups, and excellent biocompatibility [17], [18]. In particular, hyperbranched PG has been co-polymerized with cholesterol [18], PCL [19], PCL–polycitric [20], and PEG [21], for applications to intracellular delivery of paclitaxel and docetaxel [22]. In comparison to PEG, the multiple hydroxyl groups on each chain of PG can help increase the surface hydrophilicity, thereby increasing dispersion stability in the blood stream without loss of reaction sites for targeting ligand conjugations. However, hyperbranched PG potentially prevents compact and dense self-assembly of amphiphilic copolymer due to their high steric hindrance associated with a dendritic architecture, which can produce relatively large size particles [23], [24], [25], [26], [27]. Linear PG has a simple and well-defined molecular structure while still carrying abundant hydroxyl groups and flexible corona chain, holding promising properties for applications to nanocarriers.

In this work, using linear PG as a hydrophilic corona, we investigated the impact of tumor-targeting ligand multivalency on the in vivo efficacy of anti-cancer drugs encapsulated within the polymer nanocarriers. Amphiphilic block copolymers, linear PG-b-PCL and PEG-b-PCL, were synthesized and used to stabilize paclitaxel-loaded lipiodol nanoemulsions, to which folic acid (FA) was introduced as a tumor-targeting ligand. FA is a widely used ligand with a high affinity to FA receptor proteins expressed in the cytoplasmic membrane of many types of cancer cells [28]. However, its high hydrophobicity limits the number of FA that can be introduced onto the surface of nanocarriers because structural changes and dispersion instability is easily induced by their self-association by hydrophobic interactions. Both of PG-b-PCL and PEG-b-PCL also serve as emulsifiers to produce highly stable oil-in-water nanoemulsions by forming a robust semi-solid interphase between oil and water, as demonstrated in our recent works [29], [30], [31], [32]. Lipiodol, an iodinated derivative of poppy seed oil with a high iodine content (30–40 wt-%), was used as a core oil that dissolves paclitaxel [33], [34], [35], [36], [37]. Lipiodol can be also used as a contrast agent for computer tomography, although the current work is not extended to the application. The cytotoxicity, cellular uptake, in vivo bio-distribution, and in vivo tumor suppression performance of the FA-PGylated and FA-PEGylated nanoemulsions were examined to demonstrate their feasibility as tumor-targeted nanocarriers.

Section snippets

Materials

Glycidol (96%), ethyl vinyl ether (99%), p-toluene sulfonic acid monohydrate (TsOH, 98%), potassium tert-butoxide (>98%), ɛ-caprolactone (97%), tin (II) 2-ethylhexanoate (92.5–100%), oxalic acid (98%), 3-aminophenylboronic acid hydrochloride (3-APBA, 98%), FA (97%), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (EDC, 98%), N-hydroxysuccinimide (NHS), inorganic salts, and organic solvents were purchased from Sigma-Aldrich (St. Louis, Mo, USA) and used without further purification.

Results and discussion

To synthesize a linear PG block, the hydroxyl groups of glycidol were temporarily protected with ethyl vinyl ether to form ethoxyethyl glycerol ether (EEGE) because the use of unmodified glycidol results in branched PG (Fig. 1a). EEGE was then polymerized by ring-opening polymerization to produce PEEGE. The synthesized PEEGE was then used as an initiator for the synthesis of amphiphilic block copolymers through ring-opening polymerization of ε-caprolactone to produce PEEGE-b-PCL. Finally, the

Conclusion

This study clearly demonstrated the advantage of the PGylation of nanocarriers over conventional PEGylation. The PGylation allows the higher multivalency of tumor-targeting ligands of nanoemulsions, which greatly enhanced the in vivo therapeutic efficacy of the encapsulated paclitaxel. Linear PG-b-PCL diblock copolymers can efficiently stabilize lipiodol nanoemulsions by generating a robust layer at the oil/water interface. The hydrophilic PG block enables a facile conjugation of hydrophobic FA

Acknowledgements

This study was supported by a grant of the Korea Healthcare Technology R&D Project (Grant No.: HN12C0064) and a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) (Grant No.: HI14C1234), funded by the Ministry of Health & Welfare, Republic of Korea. T.H. Le Kim, J. H. Yu, and H. Jun contributed equally to this work.

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