Regular Article
Gold nanoparticle-guarded large-pore mesoporous silica nanocomposites for delivery and controlled release of cytochrome c

https://doi.org/10.1016/j.jcis.2020.12.117Get rights and content

Abstract

Efficient delivery of active proteins to specific cells and organs is one of the most important issues in medical applications. However, in most cases, proteins without appropriate carriers face numerous barriers when delivered to the target, due to their unsatisfied properties, such as poor stability, short half-life, and low membrane permeability. Herein, we have presented a large-pore mesoporous silica nanoparticle (LPMSN)-based protein delivery system. LPMSNs were obtained with ethyl acetate as a pore expander. A 2,3-dimethylmaleamic acid-containing silane coupling agent was modified on LPMSNs to provide pH-triggered charge reversal. After Cytochrome c (CC) was encapsulated in the large pores of LPMSNs, amino-terminated polyethylene glycol-modified gold nanoparticles (AuNPs) served as gateguards to cap the tunnels of LPMSNs and to avoid the leakage of CC. Above nanocomposites exhibited the capability to deliver active CC into cancer cells, charge reversal-induced protein release, as well as to initiate the apoptosis machinery of cancer cells in vitro. Importantly, the nanocomposites significantly inhibited tumor growth and extended survival rate without obvious side effects. This study provides a smart and efficient protein delivery platform with good safety profiles for efficacious tumor protein therapy in vivo.

Introduction

Proteins play critical roles in disease prevention, cancer therapies and health promotion. Compared with small-molecular drugs and genetic drugs, protein-based pharmaceuticals could directly reverse the disordered functions of cells with their advantages of definite biological function, high pharmacological activity, specificity and low side effects. Therefore, proteins have been widely used in medical applications, such as cancer treatment [1], vaccination [2] and regenerative medicine [3]. However, there are still several challenges to be addressed, such as in vivo instability caused by fast enzymatic degradation and blood clearance, and poor internalization owing to the permselectivity of plasma membrane.

In the past decades, a variety of nanocarriers have been developed to maintain the biological activity and to achieve targeted delivery of protein pharmaceuticals, such as nanogels [3], inorganic nanoparticles [6], polymers [7] and lipids [4], [5], [6], [7], [8]. Among them, mesoporous silica nanoparticle (MSN) is one of the superior inorganic nanocarriers. MSNs have been widely used for drug delivery, due to its adjustable pore size, flexible surface modification, inert chemical properties, as well as favorable biocompatibility and low biotoxicity [9], [10], [11]. However, the pore size of contemporary MSNs is up to 3 nm, which is insufficient to encapsulate protein pharmaceuticals. Recently, in order to load proteins into the mesopores, MSNs with expanded pore size larger than the protein dimensions has been developed to meet the requirement of protein delivery [12], [13], [14]. Kros et al. [15] prepared MSNs with 10 ± 1 nm of disk-shaped cavities, which provided a scaffold with high encapsulation efficiency of proteins. Kim et al. [16] reported the synthesis of extra-large pore mesoporous silica nanoparticles (XL-MSNs) to transport ovalbumin and CpG oligonucleotide into dendritic cells and evaluated their potential applications as preventive cancer vaccine.

Although the expanded pores enable MSNs to load and deliver various cargos, cargos are easy to escape from the loose and open mesopores of MSNs, resulting in undesired spontaneous drug leakage before reaching targeting cells. Recently, there have been several strategies to inhibit cargo leakage [17], [18], [19], [20], [21]. Ma et al. presented a multifunctional nanoplatform integrating PbS/CdS quantum dots and Fe3O4 nanoparticles (NPs) within large-pore MSNs (LPMSNs) enabled by simple thiol modification [22]. Salis et al. showed that MSNs with a hexagonal structure could be conjugated to two relevant proteins (e.g. bovine serum albumin and lysozyme) through Schiff-base bond [19]. Nevertheless, MSNs with large pores that are able to efficiently load and controllably release protein pharmaceuticals in the microenvironment of targeting cells are highly desired. To address these demands, we designed a gold nanoparticle (AuNP)-guarded LPMSNs (LPMSN@AuNP) with pH-triggered release of cytochrome c (CC, a crucial death regulatory protein). Ethyl acetate was used as a pore expander to obtain LPMSNs. A pH-sensitive silane coupling agent (DMMA-APTES) was grafted on the surface of LPMSNs to endow them with pH-triggered charge reversal in acidic lysosome microenvironment. Amino-terminated polyethylene glycol (PEG)-modified AuNPs (AuNP-PEG5k-NH2) were subsequently adsorbed on the surface of LPMSNs through electrostatic attraction as gate-guards to avoid the leakage of CC that can trigger apoptosis in cancer cells (Scheme 1) [23], [24]. The capability of nanocomposites to deliver proteins into cancer cells and charge reversal-induced protein release were investigated by flow cytometry and confocal laser scanning microscopy (CLSM). Furthermore, HeLa xenograft model mice were established to evaluate the antitumor effects of the nanocomposites.

Section snippets

Materials

Tetraethoxysilane (TEOS, AP), triethylamine, succinic anhydride (SA, 99%) and dimethyl sulphoxide (DMSO, AP) were purchased from Sinopharm Chemical Reagent Co., Ltd, (Shanghai, China). Hydrogen tetrachloroaurate(III) trihydrate (HAuCl4·3H2O, 99.9%), 3-aminopropyl triethoxysilane (APTES, 95%), cetyltrimethyl ammonium bromide (CTAB), fluorescein isothiocyanate (FITC, 90%) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.).

Synthesis and Characterization of nanocomposites

LPMSNs were prepared through hydrolysis of TEOS with a large amount of ethyl acetate to enlarge the mesopores and CuS NPs as seed. LPMSNs with different morphologies and pore sizes were obtained by varying the amount of ethyl acetate (Fig. S1 in Supporting Information (SI)). When a large amount of ethyl acetate (10 mL) was added, ethyl acetate formed large emulsions in the CTAB micelles, which contributed to the formation of large mesopores (Fig. 1a and b) [16], [25]. In addition, the absence

Conclusions

In summary, we have developed a novel method to prepare gold nanoparticle-guarded large-pore mesoporous silica nanocomposites for protein delivery. Compared with previously reported approaches [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], LPMSN@AuNPs nanocomposites not only provide the capability of protein encapsulation within the expanded pores, but also endow themselves intelligent response to tumor microenvironment for efficient protein delivery. Specifically, AuNPs with

CRediT authorship contribution statement

Chen Guo: Investigation, Formal analysis, Writing - original draft. Yamin Zhang: Investigation, Formal analysis, Writing - original draft. Yuce Li: Investigation. Lianbin Zhang: Investigation, Validation. Hao Jiang: Conceptualization, Supervision, Funding acquisition, Writing - review & editing. Juan Tao: Supervision, Writing - review & editing. Jintao Zhu: Conceptualization, Validation, Funding acquisition, Writing - review & editing.

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 by the National Natural Science Foundation of China (81772913 and 51525302) and the Fundamental Research Funds for the Central Universities (2172019kfyXJJS070). We thank the support from Prof. Guanxin Shen in the Immunological Laboratory of Tongji Medical College, HUST. We also thank the HUST Analytical and Testing Center for allowing us to use its facilities.

References (38)

  • R. Tang et al.

    Direct Delivery of functional proteins and enzymes to the cytosol using nanoparticle-stabilized nanocapsules

    ACS Nano

    (2013)
  • K. Dutta et al.

    Templated self-assembly of a covalent polymer network for intracellular protein delivery and traceless release

    J. Am. Chem. Soc.

    (2017)
  • M. Wang et al.

    Combinatorially designed lipid-like nanoparticles for intracellular delivery of cytotoxic protein for cancer therapy

    Angew. Chem. Int. Edit.

    (2014)
  • F.Q. Tang et al.

    Mesoporous silica nanoparticles: synthesis, biocompatibility and drug delivery

    Adv. Mater.

    (2012)
  • N.Z. Knezevic et al.

    Large pore mesoporous silica nanomaterials for application in delivery of biomolecules

    Nanoscale

    (2015)
  • H.K. Na et al.

    Efficient functional delivery of siRNA using mesoporous silica nanoparticles with ultralarge pores

    Small

    (2012)
  • X. Hong et al.

    The pore size of mesoporous silica nanoparticles regulates their antigen delivery efficiency

    Sci. Adv.

    (2020)
  • J. Tu et al.

    Mesoporous silica nanoparticles with large pores for the encapsulation and release of proteins

    ACS Appl. Mater. Interfaces

    (2016)
  • B.G. Cha et al.

    Extra-large pore mesoporous silica nanoparticles enabling co-delivery of high amounts of protein antigen and toll-like receptor 9 agonist for enhanced cancer vaccine efficacy

    ACS Central. Sci.

    (2018)
  • Cited by (19)

    • Shellac/caseinate as a composite nanocarrier for improved bioavailability of quercetin

      2023, Food Hydrocolloids for Health
      Citation Excerpt :

      The surface anionic charges along with other physico-chemical properties such as size, shape and surface properties determine the diffusion properties of the nanostructures through the mucus layer before reaching the surface of the enterocytes. Moreover, the residence of the nanoparticles is also influenced by the stability of the nanoparticles (Guo et al., 2021). Encapsulation efficiency (EE) is an important parameter to determine the efficacy of the encapsulation techniques.

    • Benefits and limitations of nanomedicine treatment of brain cancers and age-dependent neurodegenerative disorders

      2022, Seminars in Cancer Biology
      Citation Excerpt :

      The composition of nanoparticles (NPs) used in the diagnosis and treatment of brain diseases is very diverse. NPs may be composed of lipids (liposomes, micelles and lipid NPs) [21,22], polymers (dendrimers, hydrogel, polymer NPs) [23,24], carbon structures (nanotubes, fullerenes, nanodiamonds, graphene) [25–27], protein structures [28], and inorganic substances such as metals (gold, silver, iron) [29–32], silicon-based NPs [33–36], quantum dots [37,38], viral components [39–41], exosomes [42], and others (Fig. 1). Recently, there has been great progress in nanotechnology for the diagnosis and treatment of brain diseases, both in the case of cancer and neurodegenerative pathologies [12,43,44].

    • Chemiluminescence sensing of adenosine using DNA cross-linked hydrogel-capped magnetic mesoporous silica nanoparticles

      2022, Analytica Chimica Acta
      Citation Excerpt :

      The latest report about mesoporous silica and its derivatives has been widely used in controlled release systems. The system can be triggered by different stimuli, such as temperature, pH, magnetic field, etc [23–27]. Recently, a controlled release system based on mesoporous silica nanoparticles has been combined with a variety of detection technologies to establish biosensors for detecting different targets.

    • Bioencapsulation for protein delivery

      2022, Smart Nanomaterials for Bioencapsulation
    • Surface modification of silica nanoparticles by hexarhenium anionic cluster complexes for pH-sensing and staining of cell nuclei

      2021, Journal of Colloid and Interface Science
      Citation Excerpt :

      The aforesaid functional properties can be achieved by incorporation of building blocks with different functionality into SNs with a formation of hybrid SNs. Literature data represent diverse synthetic routes for synthesis of hybrid SNs [8–15], although highlighting of optimal synthetic routes for combination of sensing and cellular contrasting functions in each hybrid SN is still challenging problem. The present work is focused on optimization of hybrid SNs for joining of efficient red emission and sensing function with efficient cell internalization.

    View all citing articles on Scopus
    1

    These authors contributed equally to this paper.

    View full text