Lipid-mimicking peptide decorates erythrocyte membrane for active delivery to engrafted MDA-MB-231 breast tumour

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Abstract

Decorating the membrane surface of vesicle carriers with proteins for targeted delivery has been achieved mainly by chemical methods. In this study, we report the rational design of a lipid-mimicking peptide for biomembrane decoration without chemical conjugation. A peptide Pm45 consisting of a hydrophobic helical tail and an anionic headgroup linked with an integrin-targeting RGD moiety was manually designed. Pm45 was synthesized and characterized, which confirmed an alpha-helix at the C-terminal. Pm45 spontaneously intercalated into the lipid bilayer as illustrated by quartz crystal of microbalance with dissipation (QCM-D), a calcein leakage assay, and TEM. The intercalation was accomplished within 10 min, and the ITC results indicated that the affinity of Pm45 binding with lipids was ~100-fold greater than that of the naturally occurring cell-penetrating peptide Ib-AMP4. In vitro cellular experiments indicated that the Pm45-decorated erythrocyte vesicles specifically bound and killed integrin αvβ3-expressing MDA-MB-231 breast cancer cells. The targeting potential of Pm45-decorated erythrocyte vesicles was further evaluated in an MDA-MB-231 xenograft nude mouse model. The in vivo therapeutic effects indicated that the targeting vesicles significantly improved the therapeutic effect of encapsulated doxorubicin (DOX) compared with that of DOX or non-targeting vesicles. NIRF imaging implied that the targeting vesicles improved the pharmacokinetics of DOX in vivo and concentrated DOX in the tumour tissue at levels >50% higher than those achieved by non-targeting liposomes. This study reports a new method for liposome decoration as an alternative to chemical conjugation.

Introduction

Most of conventional chemotherapeutics for tumour therapy fail in the clinical phase due to either inability to concentrate in the target tissues or severe side effects on normal tissues caused by nonspecific drug release. Passive delivery, also termed the enhanced permeability and retention effect (EPR), contributes to the retention of nanoparticles due to the lack of normal lymphatic drainage in tumours [1]. However, the EPR effect provides only modest tumour specificity, with a 20–30% increase in delivery compared to that in normal organs [2], [3]. To further improve drug accumulation at the targets, active targeting has been proposed, as it has the potential to develop safer and more effective therapies than passive drug delivery [4].

As a well-established nanometre-scale system, liposomal nanoparticles are frequently adopted to deliver chemical drugs, genes, vaccines, and imaging agents [5], [6]. Coating or encapsulating nanoparticles with lipids offers structural features similar to those of the cellular membrane [7]. These features promote the biocompatibility of nanoparticles and present a clinically proven platform for further enhancement of nanoparticle therapeutic efficacy. Some of these nanocarriers, such as the marketed Doxil and Caelyx, are now successfully used in clinical practice, and the EPR effect has become a gold standard in the design of passive tumour-targeted systems [7], [8].

However, rapid liposomal clearance from the blood circulation by the mononuclear phagocyte system (MPS) upon intravenous administration was observed [9]. The endogenous red blood cells (RBCs) with elastic biomembranes are able to traverse capillaries to reach diseased organs and tissues [10]. Most important, RBCs possess a remarkable lifespan as long as 120 days in the circulation. The RBC-based carriers are promising for drug delivery in vivo [11], [12], [13].

Conferring active targeting potential on liposomes by decorating the surface with tumour-specific ligands combines the merits of both the passive and active delivery [4]. Amphipathic compounds capable of self-assembly are usually employed to prepare nanoparticles in conjugates with bioactive compounds [14], [15], [16], [17]. At present, there are several approaches reported for biomembrane decoration, but all these approaches share derivatization of the targeting ligands. The ligands have to be linked to an amphiphile that can be integrated into the lipid bilayer [2], [15], [18], [19], [20], [21]. The reported amphiphile molecules are normally functionalized lipid derivatives, e.g., DSPE-PEG-NHS and MonY [2], [18], [19], [20]. The amino- or carboxyl-groups from the ligands react and covalently link with the functionalized amphiphiles. Afterward, the conjugates can be incorporated into the biomembrane pre- or post-preparation. These approaches generally require simple “clicking” reactions [22], mainly catalysed by EDC/NHS without spatial specificity [23], [24], and the successful conjugation of many substrates, including proteins, aptamers, and small molecules, has been reported. Despite this great potential, however, the current approaches have limitations, particularly resulting from the heterogeneity of the conjugation products with nonspecific modification, which gives rise to analytical as well as safety and efficacy concerns. Some conjugates with heterogeneous attachment exhibited reduced clinical efficacy compared to that of the free proteins, which was at least in part linked to the nonspecific coupling chemistry [25]. As an alternative to chemical conjugation, cell-penetrating peptides might be promising for use as anchors for lipid membrane decoration due to their natural ability to intercalate in the lipid bilayer [26], [27], [28]. Ib-AMP4 representing the classic beta-sheet cell-penetrating peptide [27], could intercalate the biomembrane through a non-lytic mechanism, and was successfully applied for membrane decoration [29].

In this study, we reported and assessed a novel method of biomembrane decoration for targeting delivery as an alternative to present chemical conjugation method. A lipid-mimicking peptide was manually designed as shown in Scheme 1. A hydrophobic alpha-helical tail consisting of 32 amino acids from the C-terminal domain of human matrilin-1, a naturally occurring insoluble protein in the human body [30], was linked with a hydrophilic headgroup consisting of 4 anionic amino acids, and an RGD motif was placed in the N-terminal next to the headgroup. We applied this peptide to decorate the biomembrane of RBCs for targeting delivery. We assessed the targeting ability, therapeutic efficacy, and toxicity of the system in vitro and in vivo. The novel Pm45-RBC-Lip@DOX drug delivery system showed excellent tumour cell-targeting ability and significant antitumour efficacy, whereas toxicity to normal tissues was reduced.

Section snippets

Chemicals, medium and others

DOX was obtained from Kanion Pharmaceutical Co. Ltd. (Jiangsu, China). DMEM and RPMI 1640 and bovine serum were purchased from Gibco (Shanghai, China) for cell culture. Human breast cancer cells MBA-MD-231 and Human Embryonic Kidney 293 cells (HEK293) were purchased from the American Type Culture Collection (ATCC). Phosphate lipids (DOPC:DOPG = 3:1, mol/mol) from Avanti (Avanti Polar Lipids, Alabaster, Alabama) were dissolved in chloroform/methanol (9:1) at 2.5 mg/mL as stock solutions and used

Synthesis and characterization of the peptide Pm45

Peptide Pm45 and FITC-Pm45 synthesized by solid-phase methodology were purified by reverse-phase HPLC and characterized by mass spectrometry (Fig. S1). The CMC of the Pm45 peptide in PBS was estimated by measuring the fluorescence intensity at wavelengths of 373 and 384 nm, corresponding to the first vibronic band (I1) and the third vibronic band (I3) of pyrene, respectively. The ratio of I1 to I3 is plotted in Fig. 1A, and a sudden decrease appears at 1.25 mM, which is assumed to be the CMC

Conclusion

In conclusion, we designed the lipid-mimicking peptide Pm45 consisting of a hydrophobic helical tail and an anionic headgroup linked with the integrin-targeting moiety RGD. Pm45 showed a 100-fold greater binding/intercalation affinity with the lipid bilayer than that of the naturally occurring cell-penetrating peptide Ib-AMP4 [29]. RBC-based vesicles decorated with Pm45 at the surface specifically bound integrin αvβ3-expressing tumour cells and improved drug accumulation in the tumour tissue in

Statement of significance

1. The Lipid mimics of Pm45 peptide was consisted of an RGD motif at the anionic headgroup linked with a hydrophobic alpha-helix tail.

2. Pm45 could spontaneously intercalate into lipid biomembrane.

3. Pm45 could be used for decorating liposomes prepared from Red Blood Cells.

4. Liposomes decorated with Pm45 specially bound with αvβ3-expressing MDA-MB-231 breast cancer cells.

Contributions

X. Fan, H. Xu and G. Wu donated the blood for the experiments. H. Xu, F. Zhao and X. Fan performed the experiments. G. Wu and X. Fan conceived of the experiments. X. Fan wrote the manuscript. X. Fan, F. Zhao, C. Zhang and G. Wu revised the manuscript. J. Song and Y. Jin helped with the QCM-D experiments and discussed the results. X. Fan and G. Wu funded this study.

Declaration of Competing Interest

We declare of no conflict of interest.

Acknowledgements

This work was supported by the Fundamental Research Funds for the Central Universities (grant number 2242020K40130), the National Natural Science Foundation of China (grant numbers 81603016, 81773624), the Natural Science Foundation of Jiangsu Province (grant numbers BK20160706, BE2017746), and the National Science and Technology Major Project (grand number 2020ZX09201015).

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