Gold nanoparticles stabilize peptide-drug-conjugates for sustained targeted drug delivery to cancer cells

Abstract

Background: Peptide-drug-conjugates (PDCs) are being developed as an effective strategy to specifically deliver cytotoxic drugs to cancer cells. However one of the challenges to their successful application is the relatively low stability of peptides in the blood, liver and kidneys. Since AuNPs seem to have a longer plasma half-life than PDCs, one approach to overcoming this problem would be to conjugate the PDCs to gold nanoparticles (AuNPs), as these have demonstrated favorable physico-chemical and safety properties for drug delivery systems. We set out to test whether PEG coated-AuNPs could provide a suitable platform for the non-covalent loading of pre-formed PDCs and whether this modification would affect the bioavailability of the PDCs and their cytotoxicity toward target cancer cells.

Methods: Peptides specifically internalized by A20 murine lymphoma cells were isolated from a phage library displaying 7mer linear peptides. Peptide specificity was validated by flow cytometry and confocal microscopy. PDCs were synthesized containing a selected peptide (P4) and either chlorambucil (Chlor), melphalan (Melph) or bendamustine (Bend). Gold nanoparticles were sequentially coated with citrate, PEG-6000 and then PDC (PDC-PEG-AuNP). The physico-chemical properties of the coated particles were analyzed by electrophoresis, TEM, UV-VIS and FTIR. Stability of free and PDC-coated AuNP was determined.

Results: Biopanning of the phage library resulted in discovery of several novel peptides that internalized into A20 cells. One of these (P4) was used to synthesize PDCs containing either Chlor, Melph or Bend. All three PDCs specifically killed A20 target cells, however they had short half-lives ranging from 10.6 to 15.4 min. When coated to PEG-AuNPs, the half-lives were extended to 21.0-22.3 h. The PDC-PEG-AuNPs retained cytotoxicity towards the target cells. Moreover, whereas pre-incubation for 24 h of free PDCs almost completely abolished their cytotoxic activity, the PDC-PEG-AuNPs were still active even after 72 h pre-incubation.

Conclusions: Peptide-drug-conjugates hold potential for improving the target efficacy of chemotherapeutic drugs, however their short half-lives may limit their application. This hurdle can be overcome by easily conjugating them to gold nanoparticles. This conjugation also opens up the possibility of developing slow release formulations of targeted drug delivery systems containing PDCs.

Keywords: Gold nanoparticles; Peptide drug conjugates; Phage display; Targeted drug delivery.

Fig. 1

Flow cytometry of the binding potential of Peptide-FITC conjugates for target cells. P4-, P6- and P8-FITC conjugates were incubated at 0, 4 or 8 µM with 10⁶ A20 cells and analyzed for peptide binding. P4 and P8 peptides demonstrated a strong dose-dependent binding to the target A20 cells. These peptides were then tested for binding specificity by exposure to a series of off-target cells as shown in Fig. 3

Fig. 2

Ability of peptides to internalize into A20 cells. P4, P6 and P8-FITC conjugates were incubated with A20 cells. After several washes and counterstaining with DAPI, the cells were viewed by confocal microscopy. The P6 conjugate did not penetrate into the cells unlike P4 and P8. The FITC marker localization is extra-nuclear

Fig. 3

Flow cytometry analysis of P4 and P6 peptides binding to off-target cells. P4- and P6-FITC conjugates were incubated at the indicated concentrations with cells to ascertain the dose-binding response. The data was collected from Becton Dickson FACS Calibur cell analyzer and analyzed with Flow Jo software. Aside from the strong dose–response to A20 target cells P4 binds a small population of NB4 and HL-60 myeloid leukemic cells but not to PBMC or MOPC cells

Fig. 4

Cell growth Inhibition assay by free and conjugated drugs. Growth inhibiting effect of drug-conjugates VS free drugs was studied on a chlorambucil, b melphalan, c bendamustine. At the end of 72 h incubation period cell growth was assessed using the XTT assay: optical density (OD) was measured at 480 and 680 nm—the latter is the background absorbance. The difference between the 480 and 680 nm measurement was used to calculate the % growth inhibition (GI) in test wells compared with control cells exposed to medium alone. The results shown for each concentration point represent the mean ± standard error for two independent experiments each conducted in (n = 3)

Fig. 5

Stability of free PDCs and PDC-PEG-AuNPs. Chemostability was assessed by incubation the constructs in 0.01 M phosphate buffer solution pH 7.2 at 37 °C for different time intervals. Biostability was assessed by incubation in a mouse liver homogenate at 37 °C. At the indicated time intervals, aliquots were taken, filtered or centrifuged (respectively) and analyzed by LC–MS for PDC integrity. Graphs a and c represent results for PDCs while graphs b and d represents results for Au-PEG-PDCs. Results are expressed as mean % PDC integrity ± standard error, calculated from two independent experiments

Fig. 6

TEM images of PEG-coated gold nanoparticles. Samples were negative stained with 1% phosphotungstic acid; thereafter, images were taken. Scale bars = 200, 20 nm. a, d Naked Gold nanoparticles (negative stained with 1% phosphotungstic acid; there is no white coated around the nanoparticles (uncoated PEG6000). b, e Gold nanoparticles coated PEG-6000 (negative stained with 1% phosphotungstic acid) black color gold nanoparticles—white color PEG6000 (negative stain). c, f Gold nanoparticles coated PEG-6000 + PDC4 − chlorambucil (negative stained with 1% phosphotungstic acid)

Fig. 7

Fourier Transformed Infra-Red spectra of PEG and PDC binding to gold nanoparticles. FTIR analysis was used to characterize the chemical binding of both PEG and Peptide drug conjugate to the surface of the nanoparticle. For the chemical bond significant of each peak, please refer to the text. a A1—gold nanoparticles; A2—PEG 6000; A3—PEG-6000 coated gold nanoparticles. b B1—PEG-6000 coated gold nanoparticles; B2—gold nanoparticles coated peptide4—chlorambucil; B3—PEG-6000 coated gold nanoparticles—PDC peptide4—melphalan; B4—PEG-6000 coated gold nanoparticles—PDC peptide4—Bendamustine; B5—PDC peptide4—chlorambucil; B6—PDC peptide4– melphalan; B7—PDC peptide4—bendamustine

Fig. 8

Effect of drug or P4-PDC pre-incubation of cytotoxic activity. Free drugs, free P4-PDCs or P4-PDC-PEG-AuNPs were incubated for 24,48 or 72 h at 37 °C and then added to A20 cells for a further 72 h after which cell growth was assessed using the XTT assay: optical density (OD) was measured at 480 and 680 nm—the latter is the background absorbance. The difference between the 480 and 680 nm measurement was used to calculate the % growth inhibition (GI) in test wells compared with control cells exposed to medium alone. The results shown for each concentration point represent the mean ± standard error for two independent experiments each conducted in (n = 3). Only the results for fresh P4-PDC versus pre-incubated P4-PDC-PEG-AuNPs are shown as pre-incubation of free drugs or free PDC4 abolished their cytotoxic activity by more than 93%