Lilla
Pethő‡
a,
György
Kasza‡
*b,
Eszter
Lajkó
c,
Orsolya
Láng
c,
László
Kőhidai
c,
Béla
Iván
*b and
Gábor
Mező
*ad
aMTA-ELTE Research Group of Peptide Chemistry, Eötvös Loránd University, H-1117 Budapest, Pázmány Péter sétány 1/A, Hungary. E-mail: gmezo@elte.hu
bPolymer Chemistry Research Group, Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, H-1117 Budapest, Magyar tudósok körútja 2, Hungary. E-mail: kasza.gyorgy@ttk.hu; ivan.bela@ttk.hu
cDepartment of Genetics, Cell- and Immunobiology, Semmelweis University, H-1089 Budapest, Nagyvárad tér 4, Hungary
dEötvös Loránd University, Faculty of Science, Institute of Chemistry, H-1117 Budapest, Pázmány Péter sétány 1/A, Hungary
First published on 19th May 2020
Numerous peptide–drug conjugates have been developed over the years to enhance the specificity and selectivity of chemotherapeutic agents for tumour cells. In our present work, epidermal growth factor receptor targeting drug–peptide conjugates were prepared using GE11 and D4 peptides. To ensure the drug release, the cathepsin B labile GFLG spacer was incorporated between the targeting peptide and the drug molecule (daunomycin), which significantly increased the hydrophobicity and thereby decreased the water solubility of the conjugates. To overcome the solubility problem, drug–peptide–polymer conjugates with systematic structural variations were prepared, by linking poly(ethylene glycol) (PEG) or a well-defined amino-monofunctional hyperbranched polyglycerol (HbPG) directly or via a pentaglycine spacer to the targeting peptides. All the drug–peptide–polymer conjugates were water-soluble as confirmed by turbidimetric measurements. The results of the in vitro cell viability and cellular uptake measurements on HT-29 human colon adenocarcinoma cells proved that the HbPG and the PEG highly influenced the biological activity. The conjugation of the hydrophilic polymer resulted in the amphiphilic character of the conjugates, which led to self-aggregation and nanoparticle formation that decreased the cellular uptake above a specific aggregation concentration. On the other hand, the hydrodynamic volume and the different polymer chain topology of the linear PEG and the compact hyperbranched HbPG also played an important role in the biological activity. Therefore, in similar systems, the investigation of the colloidal properties is inevitable for the better understanding of the biological activity, which can reveal the structure–activity relationship of amphiphilic drug–peptide–polymer conjugates for efficient tumour targeting.
Numerous peptide–drug conjugates have been developed over the years, where the drug molecules were attached through an enzyme labile or pH-sensitive spacer to the targeting moiety.9 Peptide spacers sensitive to cathepsin B, a lysosomal enzyme overexpressed in different cancer cells (e.g., breast,10 pancreatic,11 lung12 and colon cancers13), enable a selective drug release in the targeted cells.14 The GFLG spacer15 is a well-known and widely used cathepsin B labile short peptide sequence, which enables the lysosomal metabolism of the bioconjugates.
However, the application of such bioconjugates is often limited by their lack of solubility. There are some strategies to enhance the hydrophilicity and water solubility of the drug delivery systems like the incorporation of ionic and/or hydrophilic amino acids (β-sulphoalanine16) or short peptide sequences (repeating Gly and Ser residues,17 polycationic or polyanionic tags18) or rather the conjugation of water-soluble polymers. In the latter case, the most applied approach is PEGylation, the conjugation of poly(ethylene glycol) (PEG).19 Several mono- and homo- or heterobifunctional PEG derivatives with various functionalities, such as amine, carboxyl, maleimide, azido, alkyne etc., have already been produced, and most of these are commercially available with different molecular weights. Based on the known disadvantages of the PEG (non-biodegradability and causinghypersensitivity),20 nowadays, nonlinear PEG-like analogues, e.g. hyperbranched polyglycerol (HbPG), are intensively investigated as biocompatible nanocarriers.21–28 HbPG has outstanding water solubility and biocompatibility; furthermore its synthesis and functionalization can be performed easily.29 Moreover, procedures to produce HbPGs by conjugation of relevant functionalities, such as amine, carboxylic, chloroacetamide and maleimide in well-defined numbers and positions, have already been developed.30–32 Such functionalized HbPGs can be utilized to synthesize novel biomaterials, drug delivery systems and/or enhance the water solubility and biocompatibility of the linked residues. Peptide–HbPG conjugates have already been synthesized by the substitution of the multiple hydroxyl groups through thiol–ene33 or azide–alkyne reactions34 or ester linkages.35 In the literature, biotinylated PEG–HbPG was conjugated in a noncovalent manner with avidin and streptavidin,36 but to the best of our knowledge, 1:
1 covalent peptide–polymer conjugates with well-defined monofunctional HbPG have not been reported so far.
During the design of the peptide-based bioconjugates, the identification of a target receptor that is overexpressed on tumour cells is essential. The epidermal growth factor receptor (EGFR) is upregulated within a high percentage (>60%) of solid tumours (e.g., lung, liver, breast, and colon cancer cells), and hence it is an attractive target for targeted tumour therapies. The EGFR belongs to the ErbB transmembrane receptor family with intrinsic tyrosine kinase activity. The ligand-receptor binding activates the kinase moiety and leads to dimerization, autophosphorylation and downstream signalling, which may result in proliferation, differentiation, enhanced cell migration and adhesion or inhibition of apoptosis.37–39
Various EGFR targeting peptides were developed and applied as drug targeting agents for cancer therapy.40–44 Phage display studies identified a dodecapeptide, GE11 (YHWYGYTPQNVI) that specifically binds to the EGFR (Kd ≈ 22 nM) as an antagonist with much lower mitogenic activity than the EGF. GE11 was shown to be able to mediate the target-specific delivery of reporter genes to EGFR overexpressing tumour cells in vitro and in vivo.45 It was also shown that GE11 was internalized efficiently without EGFR signalling activation using an alternative actin-dependent pathway.46 GE11 has already been used successfully for the delivery of nanoparticles,47,48 micelles,49 genes,50 adenoviral vectors51 and antitumour microRNAs52 as well as in photodynamic therapy.53–55
D4 (LARLLT) was identified by computer-aided design as a potential targeting peptide.56 This hexapeptide has a specific type of binding to the EGFR, to a binding pocket different from the EGF binding site. Liposomes modified with D4 were tested efficiently in in vitro and in vivo models. The D4 mediated delivery of liposomal doxorubicin was effective against EGFR overexpressing cell lines (H1299 human non-small cell lung carcinoma cells and SPCA-1 human lung adenocarcinoma) in vitro, and the conjugated liposomes were shown to gradually concentrate at the tumour site and be preferentially retained there for more than 80 hours after injection in vivo.56 D4 has also already been successfully used as a targeting moiety of a near-IR fluorescent imaging agent57 and as a photosensitizer.53,55
Since the EGFR is a potent target receptor in tumour therapy, our aim was to prepare different EGFR targeting drug–peptide conjugates to study the structure–activity relationship. These bioconjugates were synthesized using the above-mentioned peptides (GE11 and D4) as targeting moieties, and daunomycin (Dau) as a drug molecule. Daunomycin is commonly used in cancer therapy. It belongs to the family of anthracycline antibiotics that can intercalate into the DNA and interact with the minor groove while poisoning the topoisomerase IIα enzyme.58 This cytotoxic agent was attached to the N-terminal of the peptides via oxime linkage using an aminooxyacetyl linker. Based on our previous research, the oxime bond between the Dau and the aminooxyacetyl linker is stable under physiological conditions. Therefore, a proper enzyme labile spacer should be inserted into the structure between the targeting peptide and the Dau that ensures the release of an active metabolite. The GFLG spacer is proved to be cleaved by an enzyme (cathepsin B) overexpressed in the lysosomes of the tumour cells between the glycine and the phenylalanine resulting in a metabolite containing one amino acid (DauAoa–Gly–OH). This formed metabolite was confirmed to bind to the DNA similarly to the free drug and it has only slightly lower cytotoxic activity than Dau.59 However, the GFLG spacer highly increased the hydrophobicity and thereby decreased the water solubility of the conjugates. Therefore, amino-PEG and a well-defined amino-monofunctional HbPG were used to prepare drug–peptide–polymer conjugates to overcome the solubility problems. The placement of a pentaglycine (G5) spacer between the peptide and the polymers was also performed to investigate the hindrance of the polymer in receptor binding. These novel drug–peptide–polymer conjugates were tested in vitro in cytotoxicity and internalization tests on HT-29 human colon adenocarcinoma cells.
First, the drug–peptide conjugates were synthesized using GE11 (YHWYGYTPQNVI) and D4 (LARLLT) as peptide backbones. Derivatives containing the GFLG enzyme labile spacer were also prepared. This spacer was coupled to the N-termini of the sequences, and aminooxyacetic acid was applied in all cases as a functional linker for the drug conjugation. Daunomycin was conjugated to all 4 peptides (GE11 and D4 with and without GFLG) via oxime linkage, in solution under slightly acidic conditions (pH 5.1). The obtained conjugates were purified by RP-HPLC and analysed via ESI-MS (Table 1; analytical chromatograms and mass spectra are presented in the ESI,† Fig. S1–S4). As the data in the table and figures indicate, the drug–peptide conjugates were obtained in high purity and with the expected molecular weights.
Conjugate | R t (min)a | MWcalc./MWmeas. | Turbidity (100-T%)b |
---|---|---|---|
a Gradient elution (0 min 0% B, 5 min 0% B, and 50 min 90% B) in 0.1% TFA in water and 0.1% TFA in acetonitrile–water 80![]() ![]() |
|||
Dau![]() |
30.2 | 2121.9/2122.1 | 18.2 |
Dau![]() |
33.6 | 2496.3/2496.5 | 36.8 |
Dau![]() |
33.9 | 1267.1/1267.1 | 1.9 |
Dau![]() |
39.0 | 1641.5/1641.6 | 18.7 |
Since the targeting peptides contain several hydrophobic amino acids and the enzyme labile spacer further increases the hydrophobicity of the conjugates, solubility tests were performed before the in vitro assays. We found that all daunomycin–peptide conjugates were insoluble in water, therefore, stock solutions in DMSO were prepared and diluted with 9 volume excess of serum-containing cell culture medium (RPMI-1640). The turbidimetric measurements were performed directly after the preparation of the solutions by using a UV-Vis spectrophotometer and the transmittance of light of the solutions was measured at 630 nm, since the drug–peptide conjugates have no absorbance at this wavelength (Fig. S5, ESI†). Turbidity, expressed in 100-T%, is the reduction of transparency of a liquid caused by the presence of undissolved matter, so it is the opposite of clarity. The obtained turbidity results are listed in Table 1. As shown by the observed data, with the exception of the DauAoa–D4, all conjugates partially precipitated in spite of the large amount of DMSO. In addition, the turbidity steadily decreased with time, clearly indicating the slow sedimentation of the precipitated conjugates, which was visible to the naked eye as well. Because of the low solubility and the observed sedimentation of these conjugates beside the 10% (v/v) DMSO concentration, which is far above the amount that is tolerable (non-toxic) for the cells, these conjugates are not suitable for cancer treatment, hence in vitro assays were not performed. Our aim was to overcome this solubility problem, therefore, we designed water-soluble polymer containing drug–peptide–polymer conjugates. PEGylation is a well-known and straightforward method to increase the water solubility of drugs, peptides and conjugates, but its disadvantages are also already known.20 Hence, not only the commercially available amine-terminated PEG, but also a mono-amino functional hyperbranched polyglycerol (HbPG), which can be considered as a non-linear PEG-like analogue known to have outstanding water solubility and biocompatibility,30 were used to investigate the effect of the hydrophilic polymer on the solubility and the in vitro bioactivity of the conjugates. Since such polymers may cause steric hindrance in receptor–ligand binding, analogues containing a pentaglycine (G5) spacer between the polymer and the peptide were also designed. As shown in Fig. 1, for the synthesis of these drug–peptide–polymer conjugates, the GFLG spacer containing derivatives were used, because this spacer ensures an effective release of the drug,15 on the one hand. However, due to its hydrophobic character, it significantly decreases the solubility, on the other hand.
First, the GFLG spacer containing GE11 and D4 derivatives were synthesized with a carboxyl end, and these peptides were functionalized with isopropylidene protected aminooxyacetic acid61 on the N-terminal. Derivatives bearing a G5 spacer on the C-terminal were also prepared. The purified peptides were conjugated to the amine-functionalized polymers (amino-PEG or amino-HbPG) in solution through the C-terminal carboxylic groups by using a BOP coupling agent in the presence of DIPEA (Fig. 1A). The isopropylidene protecting group was cleaved with methoxyamine in 0.2 M ammonium acetate buffer (pH 5.0, 1 M solution to MeONH2) (Fig. 1B), and subsequently the free aminooxyacetylated peptide–polymer bioconjugates were reacted with daunomycin by the formation of an oxime linkage (Fig. 1C). The drug–peptide–polymer conjugates were purified by RP-HPLC and analysed using ESI-MS (Table 2; analytical chromatograms and mass spectra in the ESI,† Fig. S6–S13). As the data indicate, the drug–peptide–polymer conjugates were obtained in high purity.
Conjugate | R t (min)a | MWavg.b | Turbidity (100-T%)c |
---|---|---|---|
a Gradient elution (0 min 0% B, 5 min 0% B, 50 min 90% B) in 0.1% TFA in water and 0.1% TFA in acetonitrile–water 80![]() ![]() |
|||
Dau![]() |
35.6 | 3563.4 | 4.2 |
Dau![]() |
34.6 | 3632.4 | 3.3 |
Dau ![]() |
32.4 | 3163.3 | 2.7 |
Dau![]() |
32.2 | 3447.3 | 2.1 |
Dau![]() |
38.9 | 2757.2 | 3.7 |
Dau![]() |
39.1 | 2954.0 | 3.5 |
Dau![]() |
35.0 | 2382.2 | 2.2 |
Dau![]() |
35.4 | 2666.9 | 2.9 |
All synthesized drug–peptide–polymer conjugates were water-soluble. Nevertheless, turbidimetric measurements under the conditions used for the in vitro tests were performed to justify the positive effect of the hydrophilic polymers on the solubility of the conjugates. The conjugates were dissolved in double distilled water and diluted with serum-containing medium (RPMI-1640), then directly after the preparation of the solutions, the turbidity was measured at 630 nm. The polymer containing conjugates showed low turbidity (see Table 2), which was constant over time. This proves that the solubility problems of peptide–drug conjugates could be solved not only by the conjugation of the well-known PEG but also by HbPG. However, it is worth mentioning that the turbidity of the PEG containing conjugates was slightly higher than the HbPG-based analogues.
We presumed that HbPG can cause lower receptor binding hindrance due to the branched structure, since the hydrodynamic volume (Vh) of HbPG is lower than the Vh of PEG with similar molecular weight. To investigate and clarify the relationship between the polymer structure and the in vitro internalization ability and biological efficacy of the conjugates, different assays were performed.
The in vitro internalization of the conjugates was studied on HT-29 human colon adenocarcinoma cells by flow cytometry. In the case of an attached cell, the cell membrane can be divided into two parts: the free membrane, which is available for the conjugates, and a part of the cellular junction that might have limited accessibility to a polymer-containing conjugate.
The cells in suspension are roundish, and although the total membrane surface can be considered accessible for the conjugates, the membrane area could be smaller than that of a cell spread on a solid surface. Supposing that the internalization ability of the cells might depend on the free membrane surface, the treatment was performed on attached cells and on cells in suspension as well. The compounds were added to the cells with a final concentration of 10 μM and co-incubated for 30 min, and then the cells were analysed via flow cytometry. In all cases, one part of the cells was treated with trypsin–EDTA to investigate exclusively the amount of the internalized conjugates, since trypsin can remove the non-specifically membrane-bound compounds. As demonstrated in Fig. 2, most of the conjugates were found intracellularly. This means that with the exception of DauAoa–GFLG–GE11–G5–PEG, all drug–peptide–polymer conjugates could enter the cells. Moreover, no significant difference was observed between the uptake of the cells in suspension (Fig. 2A and B) and the attached cells (Fig. 2C and D). The polymers influenced the cellular uptake of the conjugates in a different manner, but in both groups (GE11 and D4 containing conjugates), one of the compounds was outstanding. The HT-29 cells could uptake Dau
Aoa–GFLG–GE11–PEG and Dau
Aoa–GFLG–D4–G5–HbPG to the highest extent. The G5 spacer increased the uptake of the D4-HbPG derivative, presumably because the increased distance between the globular HbPG and the very short peptide sequence provides a better receptor binding. In sharp contrast, the G5 spacer decreases the uptake of the conjugates in all other cases. The most significant difference was observed in the case of GE11–PEG, where the G5 spacer completely demolished the internalization. Probably, here the G5 spacer provides more flexibility for the linear PEG chain resulting in decreased receptor binding.
One of the best conjugates, the DauAoa–GFLG–D4–G5–HbPG was chosen for time-dependent cellular uptake studies. Cells in suspension, as well as attached cells, were treated for 15 min, 30 min or 60 min with this conjugate. Then after washing, the cells were treated with trypsin–EDTA and analysed by flow cytometry. As presented in Fig. 3, the fluorescence intensity increased under both conditions over time, though the initial uptake of the conjugate was lower in the case of the attached cells than in suspension. The effect of the prepared daunomycin–peptide–polymer conjugates on the in vitro cell viability was determined on HT-29 human colon cancer cells using the real-time cell analyser xCELLigence SP device. The cells were treated with the conjugates having different concentrations (0.1–50 μM) and monitored for 72 hours. The cell viability (%) values as the function of the conjugate concentration are presented in Fig. 4. The IC50 values were determined after 48 hours of treatment (Table 3). The parent cytotoxic agent (Dau) has a lower IC50 value (0.342 ± 0.067 μM) than the drug–peptide–polymer conjugates on the used cell line, but the application of the produced conjugates may be more beneficial based on the increased selectivity, which leads to fewer side effects. It must be noted that the used peptides, as well as the used polymers, had no toxic effect in the utilized concentration range (IC50 ≫ 50 μM). There was one outstanding conjugate from each group (Dau
Aoa–GFLG–GE11–PEG and Dau
Aoa–GFLG–D4–G5–HbPG) in the cytotoxicity measurements that correlated well with the results of the internalization studies. These conjugates were found to be the most potent ones in the viability measurement and were proved to be taken up by HT-29 cells the most effectively. Depending on the type of polymer, the incorporation of the G5 spacer had an opposite effect on the cytotoxic activity of the conjugates. In the presence of the G5 spacer, the antitumour activity of the PEGylated conjugates decreased, while the cytotoxicity of the HbPG-containing conjugates increased, especially in the case of those with the D4 targeting peptide.
![]() | ||
Fig. 4 Results of the cell viability assay performed on HT29 cells with GE11 (A) and D4 (B) containing drug–peptide–polymer conjugates. |
Conjugate | IC50 (μM) | d (nm) | PDI | cac (μM) |
---|---|---|---|---|
Dau![]() |
1.2 | 370.0 | 0.082 | 28.6 ± 4.4 |
Dau![]() |
9.1 | 171.8 | 0.137 | 9.3 ± 0.9 |
Dau![]() |
9.3 | 83.8 | 0.144 | 16.6 ± 1.6 |
Dau![]() |
6.0 | 52.3 | 0.092 | 20.2 ± 2.9 |
Dau![]() |
5.1 | 189.3 | 0.130 | 16.1 ± 1.3 |
Dau![]() |
13.5 | 157.4 | 0.076 | 56.9 ± 3.2 |
Dau![]() |
>50.0 | 76.5 | 0.057 | 6.4 ± 1.2 |
Dau![]() |
3.8 | 71.3 | 0.042 | 13.4 ± 0.5 |
We observed that some of the conjugates (DauAoa–GFLG–D4–HbPG and Dau
Aoa–GFLG–GE11–G5–PEG) could not cause complete cell death, i.e., ∼0% viability value – characteristic for cell-free culturing medium – was not achieved even at the highest concentration, since their dose–response curves reached a plateau in a lower concentration range. In our opinion, this can be explained by the different characteristics of the highly hydrophobic peptide chain and the highly hydrophilic polymer segment. Due to this amphiphilic character, self-aggregation of the conjugates may occur, which then may block the accessibility of the targeting peptide for receptor binding, thereby decreasing the efficiency of the conjugate as well. This assumption is also confirmed by the turbidity results, since the observed low turbidity values may be caused by the possible formation of nanosized aggregates. Therefore, to prove this, the aggregation dynamic light scattering (DLS) measurements were carried out. The size (d) and dispersity index (PDI) of the conjugates were compared with conjugates having 100 μM concentration and the obtained results are presented in Table 3. The size distribution curve of the Dau
Aoa–GFLG–D4–G5–HbPG conjugate as one representative sample is presented in Fig. 5 (for the DLS size distribution curves of the other conjugates, see Fig. S14–S20 in the ESI†). It is visible from these data that the formation of nanoscale aggregates was observed for each conjugate. It can be stated that the HbPG conjugates resulted in the formation of smaller aggregates compared to the PEG-containing conjugates. In addition, the size of the GE11-PEG conjugates was bigger than the D4-PEG analogues (e.g. Dau
Aoa–GFLG–GE11–PEG: d = 370 nm, and Dau
Aoa–GFLG–D4–PEG: d = 189 nm). Moreover, the size of all HbPG-containing conjugates was in the range of 50–80 nm. These can be explained by the compact highly branched structure and the lower molar hydrodynamic volume of the HbPG than that of the PEG at the same molar mass. As observed, the incorporation of the G5 spacer into the conjugates also decreases the size of the formed nanoparticles in all cases. The self-aggregation of the amphiphilic molecules takes place above a certain concentration, called the critical aggregation concentration (cac). For the determination of the cac, the intensity of scattered light was measured by DLS in a broad concentration range (0.5–500 μM) of the conjugates. The recorded scattered intensity (kcps) plotted as a function of the concentration of the Dau
Aoa–GFLG–D4–G5–HbPG conjugate, as a representative sample, is presented in Fig. 5 (right). As shown in this figure, the scattered intensity is nearly constant at lower concentrations and increases linearly at higher concentrations. The cac was defined as the intersection of the straight lines fitted to the constant and the increasing sections. The determined cac values of the conjugates are listed in Table 3 (for the scattered intensity vs. concentration curves of the other conjugates, see Fig. S14–S20 in the ESI†). As observed, the cac of all conjugates was in the range of 6–60 μM but there is a significant difference between the PEG and HbPG conjugates, which may be explained by the structural differences of the polymers. In the case of the HbPG-based conjugates, it can be stated that the G5 spacer between the targeting peptide and the polymer slightly increases the cac. Furthermore, the GE11–HbPG conjugates have higher cac values compared to the D4 analogues. This can be explained by the higher hydrophobicity of the D4 sequence and the compact structure of the HbPG that provides lower steric hindrance on the peptide part, which is primarily responsible for the self-aggregation behaviour. However, at first sight, there is no direct correlation between the structure and the determined cac in the case of the PEG-conjugates. In the case of the GE11–PEG conjugates, the incorporation of the G5 spacer between the peptide and the polymer increases the accessibility of the hydrophobic part, which leads to a lower cac value, on the one hand. On the other hand, the D4 analogue consists of only six amino acids, and in this case, the pentaglycine spacer can drastically decrease the hydrophobicity of the peptide part, which then highly increases the cac value.
The obtained DLS data contribute greatly to the understanding of the in vitro cell viability and cellular uptake results. By comparing the GE11–PEG and PEG–G5–GE11 conjugates, there is a significant difference between the cellular uptake and the IC50 values of these conjugates. Although the largest nanoparticle was formed by the GE11–PEG conjugate (370 nm, at 100 μM), its cac value is around 28 μM, which is far above the utilized concentration for the cellular uptake measurements (10 μM). In contrast, the cellular uptake of the GE11–G5–PEG conjugate was the lowest in the investigated series, and the cell viability does not follow a classical sigmoid curve (see Fig. 4A). 50% cell viability is reached around 5–10 μM but does not further decrease by increasing the concentration, i.e., the mortality is saturated. In this case, the cac is around 9 μM, therefore, above this concentration the resulting aggregates probably cover the targeting peptide part, which inhibits the receptor-mediated internalization, and thus this results in decreased toxicity. Nevertheless, the cell viability curves of the HbPG-based GE11 conjugates follow a similar pattern, and no significant differences can be observed in the calculated IC50 values and the cell internalization abilities, respectively.
In the series of D4 conjugates, the HbPG-based D4 conjugate has the worst biological efficacy. In addition, the cell viability curve of this conjugate follows a sigmoid pattern but does not reach 50% mortality even at the highest concentration. The reason for this may be that the D4–HbPG conjugate has the lowest cac (around 6 μM), which also coincides with the saturation part of the cell viability curve. This means that small-sized (∼76 nm) aggregates are already formed at low concentrations, which may enter the cells through a receptor-mediated mechanism only in the low concentration range. In contrast, D4–G5–HbPG has the lowest IC50 value (3.8 μM) and it also shows outstanding cellular uptake in the series of the D4 conjugates. Here, the G5 spacer decreases the hydrophobicity and increases the accessibility of the peptide part. Therefore, it can be concluded that in the case of a shorter targeting peptide, the HbPG is more advantageous with a short G5 spacer. Nevertheless, in the case of using PEG, the incorporation of the G5 spacer decreases the biological efficacy irrespective of the type of targeting peptide used.
Methoxypoly(ethylene glycol) amine (amino-PEG; MW: 1000) was obtained from Alfa Aesar, Thermo Fisher Scientific (Ward Hill, MA, USA). Amino-monofunctional HbPG was produced and characterized in our laboratory, as described earlier.32 Briefly, phthalimide monofunctional HbPG was synthesized by the ring-opening multibranching polymerization of freshly distilled glycidol with a phthalimide/potassium phthalimide initiating system (the monomer/initiator ratio was 15) and by using a slow monomer addition technique (feed rate: 2.5 mL h−1) at 95 °C under an inert atmosphere. The phthalimide moiety was transformed to an amine functionality through a reaction with hydrazine monohydrate in ethanol at room temperature. The number average molar mass of the used NH2-HbPG was 1230 g mol−1 (by NMR) with narrow polydispersity (Đ = 1.3) (for further characterization data see our previous article32).
For the in vitro assays, RPMI-1640 medium, trypsin–EDTA and PBS were obtained from Sigma-Aldrich Ltd (St. Louis, MO, USA), FBS was purchased from Lonza Group Ltd. (Basel, Switzerland), while penicillin/streptomycin and L-glutamine were obtained from Gibco®/Invitrogen Corporation (New York, NY, USA). TrypLe was obtained from Thermo Fisher Scientific (Waltham, MA, USA) and the E-plates were purchased from ACEA Biosciences (San Diego, CA, USA).
Peptides for the drug–peptide conjugates were synthesized on Fmoc-Rink Amide MBHA resin, and a Boc-aminooxyacetic acid (Boc-Aoa-OH) was coupled to the N-terminal. The peptides were cleaved from the resin with the 95% TFA/2.5% TIS/2.5% H2O (v/v/v) mixture and 10 eq. Aoa·1/2HCl at room temperature for 2 h. The crude products were purified by RP-HPLC and the solvent was evaporated. The pure peptides were used directly, without lyophilization for the daunomycin conjugation.
The peptides for the drug–peptide–polymer conjugates containing the GFLG spacer on the N-terminal were synthesized on Wang resin. The first amino acid derivative was always coupled with DIC and DMAP (2:
2
:
0.2 eq. to the resin capacity) for 3 hours. Isopropylidene protected aminooxyacetic acid was coupled to the N-termini of the peptides. The peptides were cleaved from the resin in 2 h with the 95% TFA/2.5% TIS/2.5% H2O (v/v/v) mixture, purified by RP-HPLC and the pure compounds were lyophilized.
The pure compounds were analysed on a Bruker Daltonics Esquire 3000+ electrospray ionization ion trap mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany), operating with continuous sample injection at a flow rate of 10 μL min−1. Samples were dissolved in 0.1% acetic acid containing the acetonitrile–water (50:
50%, v/v) eluent and mass spectra were recorded in positive ion mode in the m/z 50–2000 range.
For the experiments on cells in suspension, the TrypLE reagent was added to the monolayer cultures of HT29 cells. The effect of the TrypLE was stopped after 5 min by adding fresh medium. The cells were transferred to FACS-tubes (105 cells/FACS-tube), centrifuged and washed with fresh cell culture medium. The treatment was carried out as described above. To remove the conjugates the cellular samples were centrifuged and washed with PBS. One part of the cells was treated with trypsin–EDTA for 5 min at 37 °C and its digestion was stopped with fresh cell culture medium. After centrifugation and a washing step with PBS, the cells were analysed by a flow cytometer. The other part of the cells, after a washing step with PBS, was used directly for the measurements.
For each measurement, 10000 cells were used. The geometric mean of relative fluorescence intensity was used to quantify the membrane-bound or intracellular daunomycin-containing conjugates, and this value was adjusted with the autofluorescence of the non-treated control. For the measurement and analysis of data, CellQuest Pro software was used. There were two independent experiments with the use of two parallels and a representative result of these experiments was plotted.
CI = (Zi − Z0)/Fi |
The background measurement for 1 h time period with 1 min interval was done by using only cell culture medium. Then, the cells were seeded into the E-plate with a density of 104 cells per well and cultured for 24 h. The cells were treated with the compounds in the concentration range of 0.1–50 μM and monitored for 72 h every 20 s.
The CI change as a result of the treatment with the daunomycin–peptide–polymer conjugates was expressed as the so called normalized Cell Index, calculated by dividing the CI at a given time point by the CI at the last time point before the treatment. Viability (%), plotted on the Y-axis of Fig. 4, was calculated as the ratio of the normalized CI recorded for conjugate-treated cells and the normalized CI of the control group. To compare the cytotoxic effects of the conjugates, the IC50 values were calculated from the viability percentages obtained at 48 h for each concentration by the integrated software (RTCA 2.0) of the xCELLigence System and OriginPro 2016 (OriginLab Corporation, Northampton, MA, USA). IC50 values were determined as the concentration that resulted in 50% reduction in the normalized CI (in cell viability). Each datum represents the mathematical average of three parallels. In the case of the control wells, an adequate volume of pure cell culture medium was loaded.
According to our results, the PEG is suitable for longer targeting peptides (e.g. GE11), but the G5 spacer is not suitable irrespective of the length of the peptide because it may decrease the biological effect by increasing the flexibility of the polymer and shading of the targeting moiety. In contrast, the use of the hydrophilic hyperbranched polyglycerol (HbPG) is advantageous for short targeting peptides (e.g. D4) but only with a G5 spacer, which provides accessibility of the peptide for receptor binding and cellular uptake resulting in outstanding cytotoxicity.
Footnotes |
† Electronic supplementary information (ESI) available: Analytical chromatograms, mass spectra, DLS and critical aggregation concentration results of the conjugates; UV-Vis spectra of daunomycin. See DOI: 10.1039/d0sm00428f |
‡ Authors contributed equally. |
This journal is © The Royal Society of Chemistry 2020 |