Ligand-installed anti-VEGF genomic nanocarriers for effective gene therapy of primary and metastatic tumors
Graphical abstract
Introduction
The nucleic acid-based therapeutics have imparted important implications in treatment of a staggering spectrum of diseases, for instance, cancer [[1], [2], [3]], tissue regeneration [4], cardiovascular diseases [5], hypertension [6] and age-related macular degeneration [7], etc. Nonetheless, the genomic therapeutics are susceptible to enzymatic degradation by nucleases in biological milieu and unable to escape from endosome for entering targeted cells, which requires gene delivery vehicles [8,9]. The therapeutic genomic codes are expected to be delivered to diseased sites in pursuit of functional proteins expression in the targeted cells [10]. Compared to viral vectors, the synthetic gene carriers, especially the cationic materials formulated ones [11], such as polyethyleneimine (PEI) [12,13], dendritic polyamidoamine (PAMAM) [14,15], chitosan [16,17] and cationic liposomes [18,19], have implied their facile utilities in forming nanoscale gene condensates through electrostatic interactions, and exhibited remarkable in vitro gene expression activities in the affected cells. Despite of the tempting function of non-viral gene vehicles, the inadequate in vivo transfection activity particularly administrated through systemic route, as well as potential toxicity of the indispensable cationic compositions, impeded the clinical translation of conventional cationic gene carriers [[20], [21], [22]]. However, towards systemic administrations, the non-specific reactions during blood retention have to be minimized, as the cationic gene vehicles were easily to be cleared by immune systems, for instance, the reticuloendothelial systems (RES) [23]. Besides, recent study has also uncovered that the cationic polymer/gene complexes were likely to be accumulated and disassembled in the glomerular basement membrane (GBM) in kidney, and rapidly eliminated from the circulation [24]. Therefore, a general strategy is to shelter underneath protective shells, e.g., poly(ethylene glycol) (PEG), on to the cationic gene carriers for achieving stealth effect, preventing interaction or destabilization by other biological species, e.g., enzymes, and extending retention in blood circulation [[25], [26], [27], [28]]. Nonetheless, the PEGylation substantially reduced the internalization between PEGylated gene vehicles and cancer cells [29]. The low cellular uptake by cancer cells and scavenging by non-targeted cells, are major barriers for the PEGylated cationic gene vehicles, which may lead to therapeutic failure. In addition, the PEGylated gene vehicles are mainly passively accumulated in tumors through the enhanced permeability retention (EPR) effects [30], which could be difficult for targeting tumors that are poorly vascularized or without EPR effects, e.g., small metastatic tumors [31]. Therefore, the development of biocompatible gene delivery vehicles that are capable of specifically internalization with cancer cells for efficient gene delivery is imperative to realize the gene therapeutics towards effective and precision therapy [32].
Herein, we developed novel ligand-installed nanocarriers through a layer-by-layer preparation strategy for efficient gene delivery, and effective gene therapy of primary and metastatic tumors through the anti-VEGF approach (Scheme 1). The nanocarriers were innovated by using the biodegradable polycationic polymer, poly{N-[N′-(2-aminoethyl)-2-aminoethyl]aspartamide} [PAsp(DET)] to formulate cationic PAsp(DET)/pDNA condensates by electrostatic interactions, and then attaching the biocompatible glycosaminoglycan layer of HA on the surface of HA-NPs (Scheme 1A). The PAsp(DET) facilitates condensation of pDNA into nanoscale and endosome escape [33,34], while the HA could selectively internalize with CD44 receptors overexpressed on cancer cells and tumor vascular endothelial cells to prompt preferential intracellular delivery of pDNA payloads into cancer cells [[35], [36], [37]], and block the CD44-angiogenic signaling for pursuit of inhibited tumorigenesis [[38], [39], [40], [41]]. As VEGF is critical for tumor angiogenesis and anti-angiogenesis is an effective approach for controlling tumor growth and development, hence, it is readily envisioned that the targeted HA transportation into tumors would render anti-VEGF tumor microenvironment. Herein, the pDNA encoding soluble VEGF receptor 1 (VEGFR1) that also referred as the soluble fms-like tyrosine kinase-1 (sFlt-1), was employed as the genomic payload to construct anti-tumor therapeutics. The sFlt-1 expressed at the tumor microenvironments is assumed to exert anti-VEGF efficacy for suppression of neovasculature formation, and the sFlt-1diffused into the circulatory system forms anti-VEGF environment to actively silence the signal of angiogenetic VEGF molecules wherever VEGF was abundant, consequently eliciting inhibitory potency to both primary and metastatic tumors (Scheme 1B). The innovated nanocarriers highly decreased the toxicity of cationic polymer-based gene carriers, improved cancer cell-targeting ability, promoted the intracellular gene delivery and gene transfection efficacy, demonstrated high accumulation and deep penetration in tumors, which finally lead to effective ablation of primary and metastatic tumors with expressed high levels of sFlt-1 and anti-angiogenetic effect.
Section snippets
Materials and animals
α-Benzyl-l-aspartate N-carboxy anhydride (BLA-NCA) was purchased from Chengdu Enlai Biological Technology Co., Ltd. (Chengdu, China). Methoxy-PEG-amine (mPEG-NH2) (Mw = 10 kDa) was purchased from SinoPEG Co. (Xiamen, China). HA (Mw = 5, 37, 89, 111 and 230 kDa) was purchased from Bloomaga Freda Biopharm Co. Ltd. (Jinan, China). Diethylenetriamine (DET), N-methyl pyrrolidone (NMP), 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride (EDC) were purchased from Adamas Reagent Co. Ltd.
Preparation and characterization of nanocarriers
The PAsp(DET) homopolymer and PEG-b-PAsp(DET) block copolymer with similar number of units were successfully synthesized through n-butylamine and methoxy-PEG-NH2 initiated ring-opening polymerization of BLA-NCA, followed by aminolysis reaction with DET (Fig. S3,4) [42,43]. Firstly, the cationic pDNA/PAsp(DET) condensates (PAD-NPs) were formulated through the electrostatic interactions (Scheme 1A). The PAD-NPs with different N/P ratio (amine groups in polymer/phosphate groups in pDNA) ranging
Conclusion
In the present study, we have elaborated a tumor-targeted gene delivery system, whose surface was functionalized with anionic biocompatible glycosaminoglycan of HA. Aside from the improved biocompatibility, with reference to the specific affinity of HA to CD44 receptors overexpressed on cancerous cells, the proposed delivery systems have managed to harness HA as the targeting motif for promoted tumor accumulation and cellular internalization with targeted cancer cells. Subsequent anti-tumor
Declaration of Competing Interest
The authors declare no competing financial interest.
Acknowledgements
This research was funded by the National Key R&D Program of China (2017YFA0207900), the Recruitment Program of Global Experts (D1424002A) and the Sichuan Science and Technology Program (2018RZ0134).
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H. Zhang and 1J. Liu contributed equally to this work.