Elsevier

Biomaterials

Volume 275, August 2021, 120859
Biomaterials

Natural discoidal lipoproteins with tiny modification for tumor extracellular dissociation in antitumor chemoimmunotherapy

https://doi.org/10.1016/j.biomaterials.2021.120859Get rights and content

Abstract

Appealing cancer immunotherapy requires synchronous presentation of tumor antigens and immunoadjuvant. Herein, a “one-step” modification strategy is proposed to tinily remould endogenous discoidal high density lipoprotein (dHDL) for tumor-homing and site-specific chemoimmunotherapy. For molecular targeting therapy, lipophilic immunoadjuvant CpG oligodeoxynucleotides is conjugated to facilitate HDL-surface anchoring; and GC nucleotides provide enough reservoir for completion of doxorubicin (Dox) “sandwich”. After administration, the tiny size (~30 nm) of disc nanodrug can maneuver deeply into tumors for receptor binding and in situ structural collapse. The intracellular concentrated CpG-Dox induce potent immunogenic cell death from burst Dox liberation at acidic pH. In turn, the released antigens and CpG motifs are simultaneously recognized by dendritic cells for antigen presentation and antitumor T cell responses. Combination chemoimmunotherapy with discoidal nanodrugs performed highest tumor weight inhibitory of 93.2% and extend the median survival time at a safe level. Collectively, this study suggests that the minimalist revolution of natural dHDL particulates may provide a biomimicry nanoplatform for site-specific amplified chemoimmunotherapy.

Graphical abstract

A “one-step” modification strategy was proposed to functionalize natural discoidal HDL into a multifunctional nanoplatform for codelivery of Dox and CpG, which works for tumor cell destruction with site-specific antigen release and activates the immune cells, respectively, acting as in situ tumor vaccine.

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Introduction

Immunotherapy by educating body's own immune systems to attack tumors, has been demonstrated promising therapeutic effects in various cancers, and identified as the forefront of treatment regimens for cancer [1]. Among diverse cancer immunotherapies, cancer vaccines containing in situ tumor associated antigens and immunostimulatory agents for activation of tumor-specific immune system, have been confirmed as a key tool to eliminate tumors, providing long-term immune memory effects [2]. However, the clinical applications of cancer vaccines were limited by tumor heterogeneity and sophisticated manufacturing processes [3].

Immunogenic cell death (ICD) can transfer tumor cells into whole-cell vaccine with multiple specific T-cell epitopes and overcome tumor heterogeneity [4,5]. Tumor cells undergoing ICD are characterized by the expression of damage-associated molecular patterns (DAMPs), such as “eat me” signal calreticulin (CRT) and “danger” signal high mobility group box 1 (HMGB1) [6]. These DAMPs could recruit dendritic cells (DCs) for phagocytosis, promote the antigen presentation and elicit antitumor T cell response [7]. Doxorubicin (Dox) that has wide antitumor spectrum is one representative ICD inducer, and can improve the immunogenicity of tumor cells [8]. Despite potential, the antitumor immune response triggered by Dox are limited, mainly on account of inhibited DCs maturation in tumor sites and tumor-draining lymph, and inefficient naive T cells activation [9]. To promote the immune cascade, additional immune adjuvants are required to work together with tumor antigens for effective DCs maturation and T cells activation, stimulating a cascade of innate immune responses [10]. As a powerful immunostimulatory Toll-like receptor 9 (TLR9) agonist, CpG oligodeoxynucleotides (CpG ODN) have been in clinical trials for tumor immunotherapy [11]. Meanwhile, potentiated immunogenic stimulation requires the co-activation of DCs by antigens and adjuvants in a spatiotemporally consistent manner [12]. The ever-increasing nano-formulations have been developed to integrate various immunomodulators into one single platform for potentiating cancer-immunity cycle with enhanced safety [13].

Previous studies have demonstrated that particle geometry is crucial in controlling the behavior over different biological levels [14]. Discoidal nanostructure provided with larger surface of wide contact area, can preferentially lodge in tumor microvasculature through intense colloidal effects [15,16]. Moreover, nanoscale disc composition feels lower dislodging hemodynamic forces and can ready deposit in tumor, conferring enhanced tumor penetration and retention [14]. Despite of all, only a small number of discoidal nanostructures have been synthesized for drug delivery due to the complexity of preparation technology. The diverse natural particulates appear to be potential cancer drug delivery candidates. Ascribed to cholesterol addiction for rapid proliferation, tumor cells up-regulate the expression of high-density lipoprotein (HDL)-related scavenger receptor class B type I (SR-BI). Especially, nascent HDL possesses discoidal structure and intrinsic tumor tropism, performing as a versatile platform for delivering therapeutic agents in cancer treatment, especially immunotherapy [17,18]. Conventionally, HDL nanocarrier was reconstituted by lipids and apolipoprotein A-I (apoA-I) for drug delivery, which couldn't fully replicate the shape and biofunctions of the native HDL [19,20]. Hence, we were encouraged to explore native discoidal HDL (dHDL) with tiny modification for multidrug shielding and shuttling in cancer chemoimmunotherapy.

Herein, we introduced an endogenous dHDL nanoplatform for co-delivery of immune adjuvant CpG ODN and Dox for site-specific chemoimmunotherapy (Scheme 1). A “one-step” modification strategy was applied for nanodrug formulation. The native dHDL was isolated and purified from albumin byproduct [21], taking advantages of discoidal nanoscale particulates for tumor homing and penetration. Duplex oligonucleotides containing CpG ODN were conjugated to phospholipid DSPE (CpG-DSPE) to facilitate HDL-surface decoration; while, natural capacity of GC base pairs was easily to “sandwich” Dox, termed as dHDL/CpG-Dox. By mimicking the natural pathway, dHDL/CpG-Dox could facilitate site-specific shuttling to deep tumors and was recognized by SR-BI receptor on the tumor cells, forming a hydrophobic channel for cargo liberation. Therefore, dHDL/CpG-Dox underwent structural reorganization and released lipid component, which leaded to the extracellular accumulation of CpG-Dox for enhanced intracellular delivery. Subsequently, Dox would struggle to discharge from CpG triggered by intracellular pH downstream, contributing to immunogenic cell death for synchronous delivery of tumor-associated antigens and CpG motifs. Under the combined activation, DCs migrated into tumor-draining lymph nodes (TDLNs) for maturation and cytokines secretion, and achieved cross-presentation to promote naive T cells activation and provoke antitumor immune responses. Meanwhile, the secreted immunostimulatory cytokines could modulate tumor immunosuppressive microenvironment for enhanced immunotherapy. Collectively, tiny modification of disc HDL could achieve co-delivery of chemotherapeutics and immune adjuvant for amplified site-specific chemoimmunotherapy, and overcome the heterogeneity and complexity of cancer vaccine.

Section snippets

Materials

Human plasma precipitate Cohn's Fraction IV (F-IV was obtained from Tonrol Bio-Pharmaceutical Co., Ltd. (Anhui, China). m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and 1,2-Distearoyl-snglycero-3-phosphoethanolamine (DSPE) were bought from A.V.T. Pharmaceutical Co., Ltd. (Shanghai, China). Unmethylated CpG sequence (5′-TCGTCGTCGTTCGAACGACGT-3′) and its complementary sequence (5’-/5thiol/-ACGTCGTTCGAACGACGACGA-3′) were synthesized by Guangzhou RiboBio Co., Ltd. (Guangdong, China).

Preparation and characterization of native dHDL and dHDL/CpG-Dox

To fully replicate the shape and biological function of the native discoidal nanostructure, dHDL/CpG-Dox nanodrugs were constructed with naturally derived dHDL nanostructure by using “one-step” modification strategy. As the foundation of dHDL/CpG-Dox generation, native dHDL was extracted and purified from the albumin byproduct via ethanol precipitation and ultracentrifugation combined method, which was an operable and reproducible strategy. The endogenous dHDL demonstrated clear bands at

Conclusions

In summary, we proposed a “one-step” modification strategy for native disc HDL structure functionalization to co-deliver Dox and CpG ODN, and potentiate cancer chemoimmunotherapy. The endogenous dHDL particulates were isolated and decorated with CpG-DSPE, which provided CG base pairs for Dox insertion, thus forming homogeneous dHDL/CpG-Dox through a facile preparation procedure. By mimicking biological features of native dHDL, dHDL/CpG-Dox was designed in preferable biomedical applications for

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.

Acknowledgements

The authors gratefully acknowledge the National Natural Science Foundation of China (81872819, 82073401 and 82073795) and China Postdoctoral Science Foundation (2020TQ0359 and 2020M681790). This study was also supported by “Double First-Class” University project (CPU2018GY26). This project was also supported by the financial support from Development Funds for Priority Academic Programs in Jiangsu Higher Education Institutions and Jiangsu Qinglan Project.

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    These authors contributed equally to this work.

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