Peptide-conjugated PEGylated PAMAM as a highly affinitive nanocarrier towards HER2-overexpressing cancer cells

Abstract

Numerous effective polymers have been designed for drug and gene delivery; of these, dendrimers have been a superior nanoplatform for targeted delivery to cancerous tumor cells. Doxorubicin (DOX) is an effective first-line antineoplastic drug encapsulated by dendrimers, which may improve its biological half-life and nonspecific distribution for different cancer treatments. In this investigation, an efficient HER2-targeting nanocarrier was developed with peptide YLFFVFER (H6) functionalized PAMAM dendrimers, which could specifically transport DOX to breast cancer cells. The peptide H6 was proportionally conjugated on the peripheral side of G4 PAMAM dendrimers using bifunctional polyethylene glycol (PEG). Toxicity testing showed that the nanocarriers were nearly non-toxic, whereas the DOX-loaded nanocarriers retained high cytotoxicity for the target cells. Detection of the nanocarriers' affinity towards the HER2 protein was performed by surface plasmon resonance imaging (SPRi), and the resulting equilibrium dissociation constant (K_D value) was 7.4810 × 10⁻¹⁰ M.

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Introduction

Nanomedicine is a revolutionary approach to cancer treatment. By encapsulating targeting probes and drugs, nanoparticles can effectively improve the biodistribution, intracellular uptake and dosing efficacy.¹ To promote the delivery of biomedical entities, the size and surface characteristics of nanoparticles have been optimized to increase the blood circulation life time.² More recently, dendrimers have been used for diagnosis and treatment of cancer due to their excellent water-solubility, great potential for drug delivery and even photothermal therapy.^3–5 Poly(amidoamine) (PAMAM) dendrimers are a class of highly branched, polycationic macromolecules with precise architecture and low polydispersity. Their size can also be well-controlled in a stepwise synthesis.^6,7 The appropriate size and promising physiological properties of PAMAM allow it to enter hyperpermeable blood vessels and substantially accumulate in the tumor cells, while reducing the side effects of chemotherapeutics.^8–10 Moreover, PAMAM also improves the permeation of the drug carriers into leaky vasculature and tumor cells by prolonging their circulation time.¹¹

Peptides are attracting increasing attention as therapeutic agents due to their theoretical cell-communicating ability. Currently, peptides have been used as sensitive targeting probes in cancer therapy without affecting normal cells.¹² Peptides have been used either alone or in combination with nanomaterials via various strategies to bind to different receptors or biomarkers of the diseases. They are particularly useful as targeting probes or drug carriers in cancer diagnosis and therapy.^13,14

Peptide dendrimers and/or peptide conjugated dendrimers are becoming critical components in nanomedicine, particular since new approaches have been developed to improve production, reduce the metabolic breakdown, and enhance permeability and retention (EPR) effect.^15–18 Although peptide dendrimers are now well-accredited drug delivery vehicles for cancer therapy, in vivo application of dendrimers in small sizes remains challenging due to rapid clearance from blood circulation.^19–21 To overcome this problem, covalent conjugation of polyethylene glycol (PEG) chains to the surface of the dendrimers has been deployed to form dendrimer-based nanocarriers. This technique is well-suited to increase the molecular weight and size of the dendrimer-based drug delivery vehicles, resulting in decreased renal filtration, longer blood circulation, increased penetration into tumor by EPR effect and decreased side effects.^22–24

HER2 (human epidermal growth factor receptor 2) is a 185K_D transmembrane tyrosine kinase regulating diverse cellular functions in response to extracellular ligands. It is an important target for the development of a variety of new cancer therapies.^25,26 This receptor is located at the cell membrane and consists of two cysteine-rich extracellular ligand binding domains and a transmembrane domain.^27–29 Several ligands have been found to bind to HER2, particularly peptide H6, which was found by our group and has a calculated dissociation contact with HER2 of 6.70 × 10⁻⁷ mol L⁻¹.³⁰

In this research, we synthesized and characterized a peptide-conjugated PEGylated dendrimer, in which peptide H6 was attached to the distal end of PEGylated PAMAM dendrimer to allow targeted binding to HER2-overexpressing tumor cells. DOX was encapsulated within the nanocarrier and its targeting and imaging efficiency for breast cancer therapy was evaluated in vitro and in vivo.

Experimental section

Materials

Maleimide–PEG–NHS (M_w = 2000) was purchased from NOF Corporation (Japan). 9-Fluorenylmethoxycarbonyl (Fmoc) protected amino acids, Wang resin (loading 0.335 mmol g⁻¹) and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were purchased from GL Biochem (ShangHai, China). DOX was provided by Huafeng United Technology (Beijing, China). SKBR3 and 293T cells (human mammary gland/breast and embryonic kidney cell lines, respectively) were obtained from Cell Resource Center, Chinese Academy of Medical Sciences (China). Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS) and penicillin were purchased from GE Healthcare Life Sciences. Phosphate buffered saline (PBS) and trypsin–EDTA, 0.25% were purchased from Macgene (Beijing, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich., Ltd (Beijing, China). BALB/c mice, female, aged 6 week, body weight 15–20 g, were purchased from Vital River Laboratory (Beijing, China); testing was performed with the approval of the Ethics Committee of Peking University and followed the Guide for the Care and Use of Laboratory Animals (NRC 2011). Other reagents were purchased from Beijing Chemical Reagents (Beijing, China). All reagents were used as received and the solvents were purified according to the general procedures used before.

Synthesis of PAMAM G4

Ethylenediamine-core poly(amidoamine) (PAMAM) dendrimer was synthesized with a divergent method by repeating the Michael addition of amino groups with methyl acrylate, followed by amidation of the resulting esters with ethylenediamine.^31,32 After purification by dialysis (MWCO 3500), ¹H-NMR spectroscopy was carried out to study the structure of PAMAM G4 (Fig. S3†).

Synthesis of PAMAM–PEG–H6

PAMAM–PEG was synthesized by the reaction of the periphery NH₂ groups of the PAMAM dendrimer with NHS-activated PEGs in a molar ratio of 1 : 64 for 15 minutes in a solvent mixture of MeOH and DMSO (1 : 1) at room temperature. The primary amino groups on the surface of PAMAM were selectivity reacted with the terminal NHS groups of the bifunctional PEG derivative in modified time.^33,34 The resulting conjugate, PAMAM–PEG, was purified by a Sephadex G-50 column to remove unreacted PEGs. To synthesize the target PAMAM–PEG–peptide, the PAMAM–PEG was activated by maleimide (Mal) and further reacted with peptide H6 in a molar ratio of 1 : 16 with continuous agitation at room temperature (Scheme 1). After 30 h, the reaction mixture was dialyzed and lyophilized and PAMAM–PEG–H6 dendrimers were obtained as a white solid (Fig. 1).^35–37

Scheme 1 Synthesis of PAMAM–PEG–peptide.

Fig. 1 Schematic of the synthesis procedure of peptide-conjugated PEGylated PAMAM-DOX encapsulated.

Synthesis of peptide

In order to efficiently synthesize peptides, we used the technique known as Fmoc strategy SPPS (solid phase peptide synthesis).³⁸ Wang resin modified with Argenin (0.35 mmol g⁻¹ loading) was used as the solid phase support. All the syntheses were carried out in anhydrous DMF. 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and N-methyl morpholine (NMM) were used for coupling of Fmoc-amino acids (4 mmol). Accordingly, the reagents were dissolved with 0.4 mol L⁻¹ NMM (4 mL) in DMF and the mixture was incubated on a rotary shaker for 40 min. The deprotection step was carried out using 20 v% piperidine for 10 min on the rotary shaker to remove the Fmoc group. After elongation, the cleavage reagent (TFA (92.5 v%), water (2.5 v%), EDT (2.5 v%) and tips (2.5 v%)), was added into the vessel to remove the side chain protecting group (SP) of the residues over 2 hours. All the abovementioned experiments were carried out in the solid phase peptide synthesis vessels containing sieves. Fig. S1† shows that the sequence of peptide H6 included 8 amino acids.

¹HNMR characterization

PAMAM–PEG–H6 was characterized by ¹H-NMR (400 mHz, DMSO, Fig. S3†). The integrals of the peaks corresponding to the PEG methylene protons (3.51 ppm), PAMAM methylene protons (2.22 and 2.67 ppm) and aromatic methylene protons (6.63 and 6.91 ppm) were used to quantify the number of PEG chains and peptides per PAMAM. The calculation showed that the average ratio of PAMAM–PEG–H6 was 1 : 60 : 7.5, which might be a proper platform for active targeting drug delivery.

Size, morphology and zeta potential

TEM (Fig. 2A and B) and DLS (Fig. 2C and S4A†) measurements were carried out for further understanding of the size and morphology of the final product. The mean size increased from about 4 nm to 7 nm after PEGylation and peptide conjugation. PAMAM was positively charged (3.60 mV) and the zeta potential measurement showed no significant change after modification with PEG and peptide H6 (2.98 mV); therefore, the positive charge of the nanocarriers propelled an efficient electrostatic interaction with the negatively charged tumor cell membrane.³⁹ In addition, nanocarriers with negatively charged surfaces may experience uptake by scavenger endothelial cells in the liver.²⁴ Therefore, nanoparticles with low positive charges are suitable for drug delivery systems. The zeta potential was measured using a zeta PALS analyzer (Brookhaven Instruments Corporation, BIC) equipped with a 35 mW solid state laser (660 nm) (Fig. S4A and B†).

Fig. 2 TEM image of PAMAM G4 (A), PAMAM–PEG–peptide H6 (B) and surface zeta potential of PAMAM–PEG–peptide H6 (C).

Fluorescence spectroscopy analysis

Spectrofluorometry was used to verify the loading of DOX within the nanocarriers. Therefore, the fluorescence spectra were obtained for a group of DOX-encapsulated nanocarriers dispersed in PBS and DMEM cell culture medium, which were made from DOX solution with different concentrations (Fig. S6†). Although the encapsulation of DOX within PAMAM–PEG–H6 in DMEM medium resulted in a slight red-shift of the typical DOX absorption peak at 592 nm, there was no significant change in intensity. DOX encapsulation within PAMAM might indicate the existence of hydrogen bonds between the carbonyl groups of DOX and amino groups of the PAMAM.⁴⁰ Calculations showed that each nanocarrier could potentially encapsulate 14.1 molecules of DOX, on average.

In vitro drug release studies

In vitro DOX release studies from the PAMAM–PEG–H6/DOX were carried out using the dialysis method under acidic and neutral pH conditions.⁴¹ 10 mg of DOX-loaded nanoparticles were resuspended in separate 2 mL solutions of PBS buffer (pH 7.4) and acetate buffer (pH 5.5), and then added into the dialysis bags (MWCO 3500). The dialysis bags were suspended in different bottles containing 200 mL of the PBS buffer and acetate buffer, respectively, at 37 °C with moderate stirring. At the setting time, the samples were withdrawn and analyzed by fluorescence spectrophotometry at Ex/Em 460/510 nm. After sampling, the dialysate was immediately replaced with an equal volume of a fresh release medium. The DOX-encapsulated dendritic nanocarriers were able to release the DOX drug under both acidic and neutral pH conditions (Fig. S7†). The apparently faster release rate of each DOX-encapsulated nanocarrier at pH 5.5 compared to pH 7.4 may be due to the fact that the positively charged DOX molecules could be repelled by the protonated PAMAM branches in the acidic environment, speeding up the release of DOX from the hydrophobic dendrimer interiors.⁴²

Results and discussion

Surface plasmon resonance (SPR) spectroscopy

The curves of association and dissociation rates were recorded by real-time binding data on the chip image and analyzed using a PlexArray HT system. The dissociation constants (K_D) of dendritic peptide H6 showed more than one hundred times higher affinity towards HER2 as compared to the linear peptide, which proved the significant ability of the nanocarrier for targeting the protein (Fig. 5D).³⁰ Peptide H6 is hard to dissolve in water, however PEGylated PAMAM was water soluble; therefore, the functionalized peptide might show better capability for binding the protein in the aqueous phase.

In vitro cytotoxicity studies by MTT assay

Cytotoxicities of the free dendrimer (PEGylated dendrimer–H6), PEGylated dendrimer–H6/DOX and free DOX were evaluated by MTT viability assay against the SKBR3 and 293T cells as high and low level HER2 overexpression tumor cells, respectively. The cells were washed three times with PBS from 15 cm cell culture dishes, trypsinized, and then transferred to tubes and centrifuged. Afterwards, the cells were seeded in 96-well plates with 5.0 × 10⁴ CFU mL⁻¹ and incubated at 37 °C in humidified atmosphere with 5% CO₂ one day before the experiment.^43,44 The cells were exposed to serial dilutions of the abovementioned complexes containing DMEM and supplemented with 10% FBS and 100 U mL⁻¹ penicillin, and then incubated for one day before the viability was determined via MTT Cell Proliferation Assay. The absorbance of each group of cells was measured separately by multimode plate reader (PerkinElmer) at a wavelength of 570 nm. Cell viability was calculated by dividing the absorbance of the drug-exposed cells by the absorbance of blank cells and expressed as a percentage value. As shown in Fig. 3, no significant cytotoxicity was observed without DOX, even at high concentration of the nanocarrier. Furthermore, it is clear that DOX-encapsulated nanocarriers showed higher cytotoxicity to SKBR3 cells as compared with 293T cells.

Fig. 3 The cytotoxicity of free DOX (black band), PAMAM–PEG–H6 (red band) and PAMAM–PEG–H6/DOX (green band) against 293T cells (negative) (A) and SKBR3 cells (positive) (B) incubated for 24 h with formulated concentrations of 0.01, 0.1, 1, 10 and 100 μM.

These results indicated the efficient selectivity and higher penetration of nanocarriers into the positive cells.

In vitro cellular uptake studies

Intracellular uptake studies were performed to evaluate the transport capabilities of PAMAM–PEG–H6/DOX by confocal laser scanning microscopy (CLSM). To this end, SKBR3 as positive cells and 293T as negative cells were incubated with DMEM medium containing PAMAM–PEG–H6/DOX at a concentration of 5 μg mL⁻¹ for 12 hours to allow the cells to attach onto the cover slips. The cells were then rinsed three times with PBS and stained with Hoechst 33342 solution (20 μM) for cell nuclei imaging. The cells were next rinsed with cold PBS and imaged by a Zeiss 710 LSM confocal microscope (Carl Zeiss Microscope systems, Jena, Germany). As shown in Fig. 4, bright blue signals were observed within the cells treated with Hoechst, whereas the small red points represented DOX. Different penetration rates were observed for free DOX, PAMAM–PEG/DOX and PAMAM–PEG–H6/DOX.^45,46 These pictures clearly showed that the highest penetration was achieved using PAMAM–PEG–H6/DOX. The red points were co-localized within endosomes and lysosomes, which indicated that the nanocarriers had entered the cell through endocytosis. Comparison of PAMAM–PEG–H6/DOX with PAMAM–PEG/DOX was made to verify the affinity of peptide H6 towards the cancer cells. It was found that the cellular uptake of DOX was noticeably increased after the addition of peptide H6. Furthermore, the cellular uptake of the drug (represented by DOX fluorescence) in the HER2 high expression SKRB3 cell line was higher than the low expression 293T cell line.

Fig. 4 Confocal microscopy images of 293T cells (negative) (upper batch) and SKBR3 cells (positive) (bottom batch) treated with free DOX (A and D), PAMAM–PEG/DOX (B and E) and PAMAM–PEG–H6/DOX (C and F) incubated for 24 h.

Previous studies have reported that PAMAM-based nanoparticles could efficiently cross the cell membrane and simultaneously increase DOX-release kinetics with a higher degree of PEGylation.^47,48 In a similar study, Thomas and coworkers reported modified PAMAM conjugation with the drug, which in comparison to the free drug resulted in higher binding to the related receptor and induced cytotoxicity in tumor cells through specific cellular internalization.⁴⁹

In vivo studies

Verification of the targeting efficacy of PAMAM–PEG–H6 and, for comparison, PAMAM–PEG was performed via in vivo fluorescent imaging. Three groups were used having three female BALB/c mice bearing SKBR3 implanted tumors in each group. The tumors were induced by subcutaneous injection of 2 × 10⁶ SKBR3 cells on the right hind flanks. At ten days post-tumor implantation, the two formulations were labeled with DiR and then injected through the caudal vein to the mice, in a dose of 100 μM for each formulation. PBS was used as the control. The fluorescence images were captured by the small animal in vivo imaging system (CRI Maestro 2). The fluorescence signal of DiR in the mice was measured in the range of red light (670–900 nm) and the in vivo fluorescence imaging was accomplished at 1 h, 8 h and 24 h post-injection. As shown in Fig. 5, the time-related tumor fluorescence intensity of the peptide-conjugated nanocarrier compared with PAMAM–PEG remarkably increased after 24 h.^50–52 The enhanced efficacy indicated that the penetration and accumulation of PAMAM–PEG–H6 in tumor tissues was significantly higher than that of PAMAM–PEG. Furthermore, the capability of peptide H6 as a ligand for mediating the breast cancer targeting was confirmed.

Fig. 5 In vivo tumor targeting and therapeutic efficacy of DOX-encapsulated nanocarriers showed the time dependent accumulation of PAMAM–PEG–H6 containing DiR with 3 different groups of mice in 1 h (A), 8 h (B) and 24 h (C) after injection through caudal vein. SPRi detection of the binding affinity of H6 towards HER2 (D).

Tumor volumes and body weights were evaluated to further assess the utility of PAMAM–PEG–H6 as a carrier for DOX treatments. At ten days post-tumor implantation, the three groups of mice (each group comprising three tumor-induced mice) with a tumor volume over 200 mm³ were administered with the formulations through the caudal vein. Animals were treated with PAMAM–PEG–H6/DOX in the first group, free DOX in the second group and PBS in the third group as control, with 5 mg of each formulation per kg body weight, every 2 days for 5 doses. The tumor volumes were measured with a digital caliper and calculated using the following formula: tumor volume V (mm³) = 1/2 × length (mm) × width (mm²). Changes of body weight were measured every two days to evaluate the nanocarriers' toxicity. The tumor volumes and the mice body weights were normalized to 100% at the first day and the mice were monitored for up to 15 days for clinical symptoms. As shown in Fig. 6A, the tumor volumes from mice treated with PAMAM–PEG–H6/DOX were 27.8% ± 4.2% smaller than at the beginning of treatment. The tumor growth inhibition of free DOX was 76.9% ± 3.7%, along with significant difference in body weight, as shown in Fig. 6B. There was no remarkable change in this case with PAMAM–PEG–H6/DOX, which showed that the nanocarriers might reduce the acute toxicity of the chemotherapeutic drugs with low symptoms of toxicity compared with the control group. After all the tests, the animals were euthanized and the tumors were cut out for optical photography (Fig. 6C).

Fig. 6 The ratio of tumor growth inhibition (%) of PAMAM–PEG–H6/DOX compared with free DOX and PBS in 3 different groups of mice over 15 days (A). Body weight changes in mice over 15 days after treatment with PAMAM–PEG–H6/DOX and free DOX, with control of PBS administration (B). Photographs of tumors treated with PAMAM–PEG–H6/DOX (upper), free DOX (middle) and PBS (bottom) (C). Fluorescence images of main organs and tumors ex vivo 3 hours post-injection indicate higher selectivity and distribution of DiR-loaded nanocarriers (upper) into the tumor versus free DiR (bottom) (D).

Ex vivo imaging studies

In addition, ex vivo imaging was performed to corroborate the treatment results and to assess the distribution of DiR-loaded nanocarriers for comparison with free DiR in the tumor and the main organs. As shown in Fig. 6D, DiR-loaded PAMAM–PEG–H6 showed stronger fluorescence predominantly in the tumor, whereas, in the other treatment group (free DiR), there were no evident fluorescence differences between other organs and the tumor. Encouraged by these results, we can conclude that the use of PEGylted PAMAM modified with peptide H6 could improve the targeting of HER2 receptors expressed on cancer cells.

Conclusions

In summary, PAMAM–PEG–peptide H6 was successfully synthesized as an efficient nanocarrier for the encapsulation of chemotherapeutic drugs. The results indicated that the nanocarrier might significantly enhance the affinity of peptide H6 towards HER2 and improve its ability for tumor targeting. These capabilities have made it an effective vehicle for HER2-targeting DOX delivery in nuclear entry, particularly in vitro and also in vivo exposure.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31470049, 31270875), National High Technology Research and Development Program of China (2015AA020408), “Strategic Priority Research Program” of Chinese Academy of Sciences (XDA09040300). We also acknowledge funding from the CAS-TWAS president's fellowship for international PhD students.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: Detailed synthesis and characterization of peptide H₆, PAMAM G4 and PAMAM–PEG–H₆; fluorescence emission spectra; NMR spectra; drug release and details of SPRi detection. See DOI: 10.1039/c6ra19552k

‡ I. R. and Z. Zh. contributed equally to this work.

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