An efficient PEGylated liposomal nanocarrier containing cell-penetrating peptide and pH-sensitive hydrazone bond for enhancing tumor-targeted drug delivery

Abstract

Cell-penetrating peptides (CPPs) as small molecular transporters with abilities of cell penetrating, internalization, and endosomal escape have potential prospect in drug delivery systems. However, a bottleneck hampering their application is the poor specificity for cells. By utilizing the function of hydration shell of polyethylene glycol (PEG) and acid sensitivity of hydrazone bond, we constructed a kind of CPP-modified pH-sensitive PEGylated liposomes (CPPL) to improve the selectivity of these peptides for tumor targeting. In CPPL, CPP was directly attached to liposome surfaces via coupling with stearate (STR) to avoid the hindrance of PEG as a linker on the penetrating efficiency of CPP. A PEG derivative by conjugating PEG with STR via acid-degradable hydrazone bond (PEG2000-Hz-STR, PHS) was synthesized. High-performance liquid chromatography and flow cytometry demonstrated that PHS was stable at normal neutral conditions and PEG could be completely cleaved from liposome surface to expose CPP under acidic environments in tumor. An optimal CPP density on liposomes was screened to guaranty a maximum targeting efficiency on tumor cells as well as not being captured by normal cells that consequently lead to a long circulation in blood. In vitro and in vivo studies indicated, in 4 mol% CPP of lipid modified system, that CPP exerted higher efficiency on internalizing the liposomes into targeted subcellular compartments while remaining inactive and free from opsonins at a maximum extent in systemic circulation. The 4% CPPL as a drug delivery system will have great potential in the clinical application of anticancer drugs in future.

Keywords:

• long circulation
• pharmacokinetics
• lysosome escape
• nanocarrier
• pH-sensitive liposomes

Supplementary materials

Effect of polyethylene glycol coupled with cell-penetrating peptide in liposomes on cellular uptake

Preparation and evaluation of CPP-modified sterically stabilized liposomes

First, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]–cell-penetrating peptide (DSPE-PEG2000-CPP) conjugate was synthesized and purified according to an established procedure with some modifications.¹ CPP was reacted with a 1.2-fold molar excess of DSPE-PEG2000-NHS in dimethyl formamide with the presence of minute quantity of triethylamine. The reaction mixture was stirred overnight at 25°C and then dialyzed against deionized water using a cellulose ester membrane filter (MWCO, 3,500 Da) till unreacted CPP was removed. The product was identified by MALDI-TOF-MS (Figure S1) after being freeze-dried.

Then, CPP-modified sterically stabilized liposomes (CPP-SSL) were prepared by thin film hydration method:² soybean phosphatidylcholine (SPC):cholesterol:DSPE-PEG2000:DSPE-PEG2000-CPP (100:50:5:3) were dissolved in a mixture of chloroform:methanol (4:1 v/v), and solvent was evaporated under reduced pressure. The thin film was hydrated with phosphate-buffered saline (pH 7.0) under sonication at 37°C for 20 minutes to form CPP-SSL.

The coupling efficient (ce) of CPP on the liposomes was determined by super filter method described in the preparation of CPP-modified pH-sensitive PEGylated liposomes (CPPL) to be ~95%.

Comparison of CPPL and CPP-SSL on cellular uptake of coumarin-6

Flow cytometry was used to determine the cellular uptake of coumarin-6 loaded by CPPL and CPP-SSL in which terminal CPP density was 4%. PL served as a control group. MCF-7 cells were incubated with free serum RPMI 1640 containing 100 ng/mL coumarin-6 loaded by CPPL, CPP-SSL, and PL at pH 6.0 for 3 hours. Fluorescence intensities of each sample were measured by a flow meter based on the protocol of flow cytometry and were shown in Figure S2. Fluorescence intensities of CPP-SSL and CPPL were approximately 1.95 and 2.40 times higher than that of PL, indicating that CPP were able to penetrate cells when either directly coupled with liposomes or conjugated with the distal end of PEG in liposomes. Furthermore, lower CPP-SSL cellular uptake of coumarin-6 than CPPL demonstrated that conjugation of CPP with PEG could prevent the penetrating abilities of CPP.

Synthesis of PHS (mPEG2000-hydrazone-STR)

First, mPEG2000-Hz-PDP was synthesized by stirring mPEG2000-CHO with 3-(2-pyridyldithio) propionylhydrazide in chloroform under argon gas protection for 48 hours at room temperature. STR-SH was then dissolved in anhydrous chloroform containing mPEG2000-Hz-PDP in the presence of triethylamine. The mixture was stirred under argon gas environment for 24 hours. Chloroform was evaporated under reduced pressure, and the conjugate was purified by size exclusion gel chromatography using Sepharose CL-4B media. White powder of the product was obtained after freeze-drying.

Preparation of ^99mTc-labeled CPPL and PL

DSPE-HYNIC was first synthesized by stirring 1.28 μmol DSPE, 7 μmol S-HYNIC, and 36 μmol triethylamine in 500 μL chloroform at 55°C for 1 hour. SPC/DSPE-HYNIC/cholesterol (2/0.07/1, mol/mol/mol) and 4 mol% CPP of SPC were used to prepare HYNIC-CPPL and HYNIC-PL according to the protocol described in the preparation of CPPL. The mixture was centrifuged in an ultrafilter tube (MWCO, 30,000 Da) at 10,000× g for 30 minutes to remove unreacted HYNIC. HYNIC-CPPL and HYNIC-PL were reacted with Na ^99mTcO₄ solution containing tricine and SnCl₂ at 50°C for 30 minutes, respectively. ^99mTc-labeled liposomes were separated from free ^99mTcO₄⁻ by PD-10 column. The labeling percentage of the liposomes was approximately 16%–20% from the elution curve.

CMC determinations for PHS and DSPE-PEG2000

Since Kalyanasundaram and Thomas³ proved that the characteristic dependence of fluorescence vibrational fine structure of pyrene could be used to determine the critical micelle concentrations (CMCs) of surfactant solutions, the so-called pyrene 1:3 ratio method has become one of the most popular procedures for CMC determinations in micelle systems. For surfactants with very low CMCs (typically below 1×10⁻³ mol/L), CMC values can be obtained from the inflection point of the pyrene 1:3 ratio plots, which is the extremum value from the plot of d(I₁/I₃)/d(log c) against log c.⁴

Pyrene in acetone was added into a series of 5 mL vials, and solvents were evaporated naturally to obtain dry crystals (6×10⁻⁹ mol). A 5 mL PHS or DSPE-PEG2000 was added into each vial at a final concentration of 5.62×10⁻³–1.00×10⁻⁷ mol/L. Mixtures were sonicated for 30 minutes at room temperature to completely dissolve pyrene into the medium. Fluorescence emission of a number of surfactant solutions containing 6×10⁻⁹ mol of pyrene was recorded using an excitation wavelength of 335 nm, and the intensities I₁ and I₃ were measured at the wavelengths corresponding to the first and third vibronic bands located near 373 nm and 384 nm. The ratio I₁/I₃ is the so-called pyrene 1:3 ratio. All fluorescence measurements were carried out at 25.0°C±0.1°C with Shimadzu RF-5301PC spectrofluorometer (Japan).

Figure S3 showed I₁/I₃ ratio against surfactant concentration. CMC of PHS and DSPE-PEG2000 was 1.23×10⁻⁴ mol/L and 8.57×10⁻⁶ mol/L, respectively.

Figure S1 MALDI-TOF-MS of DSPE-PEG2000-CPP.

Notes: The molecular weight distribution indicated that CPP was conjugated with the distal end of PEG at 1:1 molar ratio to form the conjugate of DSPE-PEG-CPP7.

Abbreviations: PEG, polyethylene glycol; CPP, cell-penetrating peptide.

Figure S2 Effect of PEG coupled with CPP in liposomes on cellular uptake measured with flow cytometry analysis.

Notes: PL served as control groups. Coumarin-6 was selected as a model drug to indicate cellular uptake ability of each liposomal carrier at pH 6.0 on MCF-7 cells due to its fluorescence (λ_ex=466 nm, λ_em=504 nm [n=3]). *P<0.05, **P<0.01.

Abbreviations: PEG, polyethylene glycol; CPP, cell-penetrating peptide; PL, PEGylated liposomes; CPP-SSL, CPP-modified sterically stabilized liposomes; CPPL, CPP-modified PEGylated liposomes.

Figure S3 The relationship between I₁/I₃ of pyrene and the concentration of polymer.

Note: CMC is defined as the concentration of polymer corresponding to the inflection point in the curve. When micelle formed, the value of I1/I3 decreased significantly with the increase of concentration of polymer. CMC is a value of concentration sudden changed with I1/I3 and it was obtained by utilizing the method of partial differential.

Abbreviations: CMC, critical micelle concentration; PEG, polyethylene glycol; PHS, mPEG2000-hydrazone-stearate (mPEG2000-Hz-STR).

References

• KorenEApteAJaniATorchilinVPMultifunctional PEGylated 2C5-immunoliposomes containing pH-sensitive bonds and TAT peptide for enhanced tumor cell internalization and cytotoxicityJ Control Release2012160226427322182771
   PubMed Web of Science ®Google Scholar
• ChiuSJLiuSPerrottiDMarcucciGLeeRJEfficient delivery of a Bcl-2-specific antisense oligodeoxyribonucleotide (G3139) via transferrin receptor-targeted liposomesJ Control Release2006112219920716564596
   PubMed Web of Science ®Google Scholar
• KalyanasundaramKThomasJKEnvironmental effects on vibronic band intensities in pyrene monomer fluorescence and their application in studies of micellar systemsJ Am Chem Soc197799720392044
   Web of Science ®Google Scholar
• AguiarJCarpenaPMolina-BolívarJACarnero RuizCOn the determination of the critical micelle concentration by the pyrene 1:3 ratio methodJ Colloid Interface Sci20032581116122
   Web of Science ®Google Scholar

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant No 81202469). The authors would like to thank Professor Hongmei Jia and Dr Jie Lu from Beijing Normal University for generously providing HYNIC and SnCl₂-Tricine kits for labeling liposomes with ^99mTc.

Disclosure

The authors report no conflicts of interest in this work.