Microfluidic self-assembly of a combinatorial library of single- and dual-ligand liposomes for in vitro and in vivo tumor targeting

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Abstract

Precise engineering of nanoparticles with systematically varied properties (size, charge surface properties, targeting ligands, etc.) remains a challenge, limiting the effective optimization of nanoparticles for particular applications. Herein we report a single-step microfluidic combinatorial approach for producing a library of single and dual-ligand liposomes with systematically-varied properties including size, zeta potential, targeting ligand, ligand density, and ligand ratio. A targeting ligand folic acid and a cell penetrating peptide TAT were employed to achieve the optimal synergistic targeting effect. In 2D cell monolayer models, the single-ligand folic acid modified liposome didn’t show any enhanced cellular uptake, while the incorporation of TAT peptide “switched on” the function of folic acid, and induced significant elevated cellular uptake compared to the single ligand modified liposomes, showing a strong synergistic targeting effect. The folic acid and TAT peptide dual-ligand liposome also demonstrated enhanced tumor penetration as observed using 3D tumor spheroid models. The in vivo study further confirmed the improved tumor targeting and longer tumor retention (up to 72 h) of the dual-ligand liposomes. Our work not only proved the versatility of this microfluidic combinatorial approach in producing libraries of multifunctional liposomes with controlled properties but also revealed the great potential of the optimized liposome formulation for synergistic targeting effects.

Introduction

Liposomes are spherical vesicles with an amphiphilic lipid bilayer enclosing an aqueous core [1], [2]. Due to the advantages such as good biocompatibility and good stability, liposomes have gained an intense interest in various fields ranging from vaccine, drug delivery to diagnostics and imaging. From the first FDA-approved nano-drug Doxil [3] to those latest liposome formulations under clinical development [4], [5], we have witnessed significant advances in developing lipid-based drug delivery systems for cancer diagnosis and treatment over the past decades. Recently, a lot of effort has been made to develop multifunctional liposomes [6], [7], [8]. Through a synergistic manner, these liposomes might address two or more challenges at the same time, such as overcoming multiple barriers to deliver drugs to specific sites, enhancing targeting specificity or increasing circulation time and tumor retention capability.

To achieve desired biological functions, liposomes are usually prepared with some specific ligands. Their biological function can be further enhanced by incorporating two or more ligands at the same time, such as using two targeting ligands or using an active targeting ligand and a cell-penetrating peptide. Ying et al. [9] developed dual-ligand liposomes using two active targeting ligands for transporting the drug across the blood-brain barrier and then targeting brain glioma. Takara et al. [10] used a specific ligand NGR and a cell-penetrating peptide oligoarginine to improve the cellular uptake efficiency through a synergistic effect. These dual-ligand liposomes [11] are promising candidates to achieve advanced properties and functions such as triggered release, better tumor targeting, and more efficient drug delivery. However, traditional methods for preparing liposomes are mainly based on bulk methods such as thin-film hydration, freeze-drying, detergent depletion and alcohol injection [12]. These methods always require post-processing steps to homogenize the size, thus often resulting in big batch-to-batch variations and poor quality control. To prepare more complex multifunctional liposomes, traditional methods might require further steps to conjugate or incorporate functional groups into the liposomes [13], [14], [15], thus leading to increased difficulties in controlling the properties and reproducibility of such liposomes, as well as the associated high manufacture cost and time consumed. In addition, as there are so many factors which can affect the in vitro and in vivo properties of multifunctional liposomes, it is essential to prepare a library of liposomes with varied compositions, thus allowing the systematic screening and evaluation. Therefore, it is critical to developing a new platform technology for investigating multifunctional liposomes in a simple, efficient, and reproducible way.

Microfluidic technology is a newly emerging method for the preparation of various nanoparticles due to its better manipulation of the synthesis process [16], [17], [18]. To prepare multifunctional liposomes in a single step, a microfluidic hydrodynamic flow focusing (HFF) approach has been developed [19], [20]. This method can provide a well-controlled mixing of the organic solvent which contains the lipids and the aqueous buffer in the microfluidic device, thus precisely controlling the properties of the liposomes such as size, charge, and surface chemistry [21]. The method also allows combinatorial synthesis of libraries of liposomes with systematically-varied properties, which has been considered as an effective way to discover new drugs through high-throughput screening in the pharmaceutical industry. Moreover, this “micro-” preparation process can be easily scaled-up by using high aspect ratio or parallel integrated microfluidic devices [22], [23], which is suitable for massive clinical applications and industrial production.

In this study, we used the hydrodynamic flow focusing method to generate a library of liposomes with varied properties, including different particle size, single ligand, dual-ligand and different ligand densities in a one-step manner. We employed a targeting ligand folic acid (FA) for active tumor targeting and a cell penetrating peptide (TAT) for efficient cell membrane translocation of the liposomes. Their biological functions were evaluated using two-dimensional (2D) cell monolayer, three-dimensional (3D) tumor spheroid models, and a tumor-bearing mouse model. The work offers a new strategy for preparing libraries of multifunctional liposomes, and the screening of them via various models allows the identification of the best formulation with the optimal biological functions. This strategy could pave the way for the liposome-based pharmaceutical applications.

Section snippets

Materials

TAT peptide with a terminated cysteine (Cys-TAT, CYGRKKRRQRRR, MW 1663) was synthesized by GenScript Corporation (Piscataway, NJ, USA). 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2000) were obtained from Avanti Polar Lipids (Alabaster, AL, USA). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000] (DSPE-PEG2000-Folate) and 1,2-distearoyl-sn

Results and discussion

We used a one-step process to prepare liposome libraries of PEGylated liposomes, single-ligand liposomes (FA modified liposomes and TAT modified liposomes), and dual-ligand liposomes (FA and TAT co-modified liposomes), with different size, the same charge, and different ligand density. We used 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol as the basic liposome components and DSPE-PEG2000 as the PEGylated lipid, DSPE-PEG2000-FA and DSPE-PEG2000-TAT were incorporated to make

Conclusion

A library of single-ligand and dual-ligand liposomes incorporating an active targeting ligand FA or/and a cell penetrating peptide TAT are synthesized using a combinatorial one-step microfluidic method. By adjusting the key parameter of the microfluidic method – flow rate ratio (FRR), the liposome size can be precisely controlled while the surface properties such as zeta-potential and ligand density remaining constant. The dual-ligand liposomes show increased monolayer cellular uptake and

Acknowledgement

This project is supported by the Australian Research Council (ARC) Future Fellowship Project (FT140100726). Rui acknowledges Ph.D. scholarships from the University of Queensland. This work was performed in part at the Queensland node of the Australian National Fabrication Facility, a company established under the National Collaborative Research Infrastructure Strategy to provide nano and micro-fabrication facilities for Australia’s researchers. The authors acknowledge the facilities, and the

References (46)

  • J. Sudimack et al.

    Targeted drug delivery via the folate receptor

    Adv. Drug Deliv. Rev.

    (2000)
  • V.P. Torchilin

    Tat peptide-mediated intracellular delivery of pharmaceutical nanocarriers

    Adv. Drug Deliv. Rev.

    (2008)
  • Q.Y. Zhang et al.

    A pH-responsive alpha-helical cell penetrating peptide-mediated liposomal delivery system

    Biomaterials

    (2013)
  • Y.Y. Liu et al.

    Paclitaxel loaded liposomes decorated with a multifunctional tandem peptide for glioma targeting

    Biomaterials

    (2014)
  • A. Nagayasu et al.

    The size of liposomes: a factor which affects their targeting efficiency to tumors and therapeutic activity of liposomal antitumor drugs

    Adv. Drug Deliv. Rev.

    (1999)
  • J. Leng et al.

    Kinetics of the micelle-to-vesicle transition: Aqueous lecithin-bile salt mixtures

    Biophys. J.

    (2003)
  • C.B. He et al.

    Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles

    Biomaterials

    (2010)
  • R. Ran et al.

    Microfluidic synthesis of multifunctional liposomes for tumour targeting

    Colloid Surf. B

    (2016)
  • Y. Huang et al.

    Curb challenges of the “Trojan Horse” approach: smart strategies in achieving effective yet safe cell-penetrating peptide-based drug delivery

    Adv. Drug Deliv. Rev.

    (2013)
  • R.A. Petros et al.

    Strategies in the design of nanoparticles for therapeutic applications

    Nat. Rev. Drug Discov.

    (2010)
  • D. Bobo et al.

    Nanoparticle-based medicines: A review of FDA-approved materials and clinical trials to date

    Pharmaceut. Res.

    (2016)
  • V.P. Torchilin

    Multifunctional, stimuli-sensitive nanoparticulate systems for drug delivery

    Nat. Rev. Drug Discov.

    (2014)
  • F. Perche et al.

    Recent trends in multifunctional liposomal nanocarriers for enhanced tumor targeting

    J. Drug Deliv.

    (2013)
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