Folate-conjugated stealth archaeosomes for the targeted delivery of novel antitumoral peptides

Abstract

In this work, novel archaeosomes based on Egg-PC and a mixture of PEGylated archaeal tetraether lipids were investigated as nanocarriers for in vitro delivery of an original anticancer peptide. With the aim to develop site-specific drug targeting, a tetraether equipped with a folate ligand at the PEG₅₀₀₀ terminal end (FA–PEG₅₀₀₀–tetraether) was synthesized in order to bind to folate receptors (over)expressed on the tumor cell surfaces. The original peptide A1 and its inactive analogue A1Yala (17 amino acids) were encapsulated into Egg-PC vesicles incorporating FA–PEG₅₀₀₀–tetraether and/or PEG₂₀₀₀–tetraether lipids in order to evaluate the in vitro anticancer activity of A1-loaded archaeosomes. Results showed a particular behaviour when A1 was encapsulated into the folate-equipped archaeosomes particularly during the first hour of incubation.

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1. Introduction

Tumour specific drug delivery has become increasingly interesting in cancer therapy, as the use of chemo-therapeutics is often limited due to severe side effects.¹ Indeed, these unwanted toxic effects impose dose reduction, treatment delay, or discontinuance of therapy that can dramatically decrease the therapeutic index of the cytotoxic agents. Amongst bioactive molecules, peptides exhibit great potential as anticancer drugs² but their use is strongly affected by their rapid elimination from the circulation, enzymatic degradation, uptake by the reticuloendothelial system (RES) and accumulation in non-targeted organs and tissues. Peptide entrapment into site-specific delivery nanovectors equipped with specific ligands for cancer cells can represent an efficient strategy to enhance the influx and retention of the drug in tumour cells. Several nanosystems have been used to encapsulate therapeutic peptides, such as polymer nanoparticles, liposomes, niosomes, and hydrogels.³ Over the last decade, we and others focused attention on the development of an innovative family of liposomes, termed archaeosomes made with one or more of either the ether lipids found in archaea or synthetic archaeal lipid analogues.⁴ These atypical vesicles demonstrate remarkable stabilities in several conditions (oxidative stress, high temperature, acidic or alkaline pH, serum media, action of enzymes) notably when membrane-spanning tetraether-type bipolar lipids were incorporated into the bilayer membrane.⁵ Therefore, because of their biocompatibility and higher in vivo stability, archaeosomes have been investigated for the delivery of peptides and proteins, including the oral and topical administration routes.⁶

Previous studies achieved in our group have clearly shown the interest in developing synthetic analogues of natural archaeal lipids for their uses in archaeosome formulations as efficient drug/gene delivery systems.⁷ In particular, diether and tetraether lipids functionalised with a short poly(ethylene glycol) chain (PEG₅₇₀) were equipped with a folate group with a view to in vitro transfection mediated by cell ligand–receptor interactions.⁸ Our next objective was to evaluate the potential of a next generation of archaeosomes that combined longevity in blood circulation and active targetability towards tumour cells. This new formulation (1) is composed by 90 wt% of a classical lipid, Egg-PC, 5 wt% of a PEG₂₀₀₀–tetraether and 5 wt% of a FA–PEG₅₀₀₀–tetraether possessing a folic acid (FA) residue at the terminal end of a PEG₅₀₀₀ chain (Fig. 1). Indeed, previous studies relative to the use of archaeosomes as gene delivery systems showed that the incorporation of 5 wt% to 10 wt% of tetraether archaeal lipids into bilayered vesicles led to the most efficient in vitro gene transfection properties.⁹ The coating of archaeosome surface with hydrophilic PEG polymers of 2000 and 5000 Da will delay the elimination process from the blood stream by endowing them with stealth properties. Indeed, the PEG stabilizing effect sterically inhibits both hydrophobic and electrostatic interactions of a variety of blood components at the nanocarrier surface. The folate ligand was grafted to the tetraether lipid possessing the longer PEG₅₀₀₀ chain so as to ensure its availability toward folate receptor (FR) over-expressed in many tumour cells.¹⁰

Fig. 1 Structure of the folate-conjugated stealth archaeosome.

The drug considered in this work is a new peptide A1 (17 amino acids, 2302 Da, RRKYGRDFLLRFRYIRS) and its inactive analogue A1Yala (17 amino acids, 2125 Da, RRKAGRDFAARFRYIRS). We previously mentioned that the last five amino acids constitute a penetratin domain that was necessary for the peptide effect to provoke cell death.¹¹ This A1 peptide exhibited high and rapid antitumoral activities on different cell lines representative of the main human cancers.¹¹ However, medicinal application of this peptide requires developing an original vectorisation approach in order to protect it in biological environment and to improve its selectivity toward targeted cells. Within this context, we aimed at evaluating the in vitro cytotoxicity of encapsulated peptides in folate-functionalized archaeosomes and to study the internalization-type of the formulated system into tumour cells.

2. Results and discussion

2.1. Synthesis of PEGylated tetraether lipids

The chemical synthesis of MeO–PEG₂₀₀₀–tetraether–OH 9 (ref. 9) (Fig. 1) was previously described but not that of FA–PEG₅₀₀₀–tetraether. With regard to the synthesis of FA–PEG₅₀₀₀–tetraether–OH 8, a first strategy based on the introduction of the folate moiety 1 into a PEGylated tetraether possessing a terminal amine, was ineffective whatever the coupling conditions used (TBTU/DIEA; EDCI/Pyr; PyBop/DIEA; DCC/Pyr). An alternative synthetic route was then envisaged using first a PEGylation step of FA followed by coupling the resulting PEGylated FA 3 with a tetraether lipid (Scheme 1). Compound 3 was prepared by activating the commercially available folic acid 1 with NHS/DCC then the dissymmetrical H₂N–PEG₅₀₀₀–NHBoc 2 was introduced to the mixture to obtain the desired product. Purification of FA–PEG₅₀₀₀–NHBoc 3 by dialysis against DMSO (3500 D cell-membrane cutoff) allowed complete removal of free FA molecules and furnished a mixture of regioisomers resulting from the coupling reaction at the α or γ position of FA, in addition to unreacted H₂N–PEG₅₀₀₀–NHBOC 2 (94% yield). After quantitative deprotection of the amino groups under acidic conditions, a yellow solid mixture was isolated as a 46 : 38 : 16 mixture of the two regioisomers γFA–PEG₅₀₀₀–NH₂ 4γ and αFA–PEG₅₀₀₀–NH₂ 4α and H₂N–PEG₅₀₀₀–NH₂ 5 determined by NMR studies. Indeed, it has been shown that the γ-carboxylic acid possesses a higher reactivity than the α-carboxylic acid.¹² Functionalisation of HOOC–tetraether–OAc 6 (ref. 9) by PEGylated folate 4 was then performed by applying the same strategy described above. Firstly, carboxylated lipid 6 was activated using DCC/NHS coupling system. Secondly, the terminal amine of the PEGylated folate 4 was added. Under these conditions and after dialysis against DMSO/CHCl₃ (removal of unreacted tetraether 6), γ/αFA–PEG₅₀₀₀–tetraether–OAc 7γ/α was deacetylated by sodium methoxide to afford a final mixture comprising a 45 : 35 : 17 : 3 molar ratio of γFA–PEG₅₀₀₀–tetraether–OH 8γ, αFA–PEG₅₀₀₀–tetraether–OH 8α, FA–PEG₅₀₀₀–NH₂ 4 and H₂N–PEG₅₀₀₀–NH₂ 5. Thus, it should be noted that as described by several authors the presence of a mixture of regioisomers α and γ in the final product 8 is not disturbing for the biological assays.¹³

Scheme 1 Synthetic route of FA–PEG₅₀₀₀–tetraether–OH 8.

2.2. Preparation and characterization of archaeosomes based on PEGylated tetraether lipids

FA-conjugated archaeosomal formulations have been prepared using the classical lipid film hydration method followed by vesicle size reduction under sonication (3 times 5 min with intervals of 5 min using a Fisher scientific sonication bath at 80 kHz) to provide vesicles with a total lipid concentration of 1 mg mL⁻¹. Particle mean diameters and polydispersity index were measured by dynamic light scattering (DLS) and revealed that archaeosome diameters were around 80 nm with a polydispersity index of 0.30. Cryo-TEM was also employed to investigate the morphology of the formed empty vesicles and the resulting data confirmed the DLS analyses. Indeed, the image of Fig. 2 shows the presence of well-shaped and dispersed spherical empty archaeosomes. Moreover, the external dark circle on the spherical-objects evidences the lipid layer surrounding an internal aqueous volume of the vesicles. Also, these nano-systems possess a diameter comprised between 20 and 200 nm. It is noteworthy that no phase segregation has been observed proving the relative homogeneity of the preparation. Nevertheless, the P.I. value obtained by DLS is not totally in accordance with the vesicle diameters observed in cryo-TEM experiments. This slight difference could be explained by the fact that these two methods do not measure exactly the same physico-chemical parameter: with DLS the hydrodynamic radius of the particle is determined whereas TEM provides an estimation of the projected area diameter without considering the hydration layer. The lyophilization of archaeosomal formulations was next envisaged to provide a simple and reproducible technique for the preparation of peptide-loaded vesicles. Indeed, the archaeosomes can be reconstituted just before the encapsulation step by rehydration of the lyophilized products by the aqueous solution containing the peptide drugs.

Fig. 2 Cryo-TEM photo of empty Egg-PC/PEG₂₀₀₀–tetraether/FA–PEG₅₀₀₀–tetraether (90 : 5 : 5 wt%) archaeosomes. Bar is 50 nm.

A1 and A1Yala peptide solutions were prepared in milliQ water at a 1% molar ratio of DMSO to facilitate their solubilisation. However, DMSO like any amphiphilic solvent is known for its disruptive effect toward lipid vesicles. For this reason and in order to select the appropriate encapsulation method, we studied at the first place the influence of DMSO on the physicochemical properties of nano-formulated vesicles. Thus, four identical freeze-dried archaeosomal formulations (see experimental part 3.3.1.) were hydrated in milliQ water in the presence of 0%, 1%, 10% and 20% (molar ratios) of DMSO respectively. After the sonication process (3 times or 7 times 5 min with intervals of 5 min) and one hour rest, particle mean diameters and polydispersity index (P.I.) were measured by Dynamic Light Scattering (DLS). The obtained results are summarized in Fig. 3 and clearly show a non-significant influence of 1% DMSO on the average diameter of vesicles and P.I. of the nanoparticle formulations. Consequently, an encapsulation technique dedicated to hydrophilic substrates proves to be the most appropriate way in our case. It is worth to be noted that the 20% DMSO formulation displayed a good average diameter and P.I. after 35 min sonication. Indeed, it has been shown a beneficial effect of DMSO at low concentrations on the average diameter of vesicles which could be due to an internalization of DMSO between water and the hydrophilic head-groups of the lipid membrane.¹⁴

Fig. 3 Effect of DMSO on average diameter (nm) and polydispersity index (P.I.) of archaeosome formulations. Points = P.I. values obtained after 15 min (in red) or 35 min (in black) sonication. Filled rectangles = average diameter values obtained after 15 min (in red) or 35 min (in black) sonication.

2.3. Encapsulation of peptides

Thereafter, a new analytical LC-MS method was developed in order to quantify the concentration of the encapsulated peptide in the archaeosomes. Firstly, HPLC separation conditions were promoted to isolate the desired peptide from the lipid mixture (Egg PC, FA–PEG₅₀₀₀–tetraether–OH 8 and MeO–PEG₂₀₀₀–tetraether–OH 9). Secondly, a calibration curve was expanded for a peptide concentration ranging from 5 to 20 μM by linearization of the area of the LC-MS chromatogram in function of the concentration of the sample. The correlation coefficients of all calibration curves were greater than 0.99 indicating a high linearity of the graphs. In order to ensure the reproducibly and the accuracy of the method, the same sample of each peptide was injected 6 times to control the values of the measured area of the chromatogram (see ESI†). Also in the interests of accuracy, we opted to realize the calibration curve experiments and to quantify the desired peptide on the same day.

Subsequently, the peptide loading procedure was performed using the hydration method of the freeze-dried vesicles as described in the experimental part. Initially, 1 mg of empty lyophilized archaeosomes was hydrated with 1 mL of the desired peptide (100 μM) and left at 4 °C for 5 hours. After a sonication step and an elimination process of the unencapsulated peptide by filtration through 50 000 NMWL pore filters by ultracentrifugation, the encapsulation efficiency determined using the quantification method described above was found to be modest for the two peptides (18% for A1 and 9% for A1Yala). For that reason, in the next step which consisted in preparing solutions for biological assays, the same procedure was reproduced but a hydration time of 23 h instead of 5 h was envisaged. After removal of the non-encapsulated peptides by ultrafiltration, the supernatants were recovered with a PBS buffer solution. Thus, 18 mL of A1 formulation and 4.84 mL of A1Yala were prepared at a concentration of 100 μM. In addition, 6.86 mL of empty vesicles were also performed for negative biological controls. Interestingly, the encapsulation level has increased for both formulations (A1 30% and A1Yala 14% respectively). For each experiment, the concentration of the peptide was controlled in the filtrate and the pellet fractions. The cumulated values found in the two fractions were very close to that of the initial concentration of the peptides (peptide A1-loaded formulation: 30% and 70% of peptide in pellet and filtrate fractions respectively; peptide A1Yala-loaded formulation: 14% and 79% of peptide in pellet and filtrate fractions respectively).

Evaluation of the physicochemical characteristics of these peptide-loaded formulations was performed through the determination of particle mean diameters and polydispersity index by dynamic light scattering (DLS). The results obtained for all preparations are summarized in Table 1. The average diameters of nano-objects increased respectively with the degree of encapsulation and the peptide nature. The presence of aggregates of loaded-archaeosomes, or the specific location of A1 compared to A1Yala within vesicles could justify the larger average diameter (473 nm) of A1-containing archaeosomes compared to A1Yala-based vesicles (156 nm). Another possible explanation could result from the presence of larger amounts of encapsulated A1 peptides that would impact/modify more deeply the archaeosome structure. This second justification seems more reasonable, seeing the similarity in chemical structure between the two peptides. Indeed, A1 is an non-amphipathic peptide that displays similar charge and hydrophobicity profiles as its inactive counterpart A1Yala.^11a The size of peptide-loaded archaeosomes is a crucial parameter for in vitro/in vivo applications since it plays a key role on the internalization mechanism. Indeed, a size slightly below 500 nm should prevent a clathrin-mediated pathway of endocytosis that shows an upper limit for internalization of approx. 200 nm.¹⁵ Conversely, these particles may enter cells preferentially along the pathway of caveolae-mediated endocytosis.¹⁵ The DSC measurements suggested the presence of Large Unilamellar Vesicles (LUV)-type or a mixture of LUVs and Small Unilamellar Vesicles (SUV) that could be confirmed through further cryo-TEM investigations of peptide-loaded formulations.

Table 1 Characteristics of peptide-loaded formulations prepared for the biological assays^a

Formulation	Encapsulation efficiency (%)	Peptide concentration (μM)	Total volume (mL)	Cm peptide (μg mL^−1)	Cm lipid (mg mL^−1)	Mass ratio peptide/lipid (%)	Average diameter (nm)	P.I.
a Cm: mass concentration; P.I.: polydispersity index.
Peptide A1	30	100	9.18	230.1	3.35	6.8	473	0.21
Peptide A1Yala	14		4.84	210.5	7	3	156	0.25
Empty vesicles	—	—	6.86	—	11	—	116	0.25

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2.4. In vitro biological assays

In vitro biological assays were then carried out on HeLa cell line highly expressing folate receptors¹⁶ in order to study the importance of encapsulating A1 in the folated archaeosomes during the vectorization step. As described previously,¹¹ A1 exhibits a drastic and rapid cytotoxic activity on cancer cell lines. As expected, we observed an important cell mortality close to 80% after only 20 minutes of incubation (Fig. 4). Biological activity of peptide–archaeosome formulations was tested at 1.7 mg mL⁻¹ of lipids corresponding to 50 μM of A1 or 24.4 μM of A1Yala and at 1.1 mg mL⁻¹ of lipids corresponding to 32.5 μM of A1 or 15.5 μM of A1Yala. Empty archaeosomes showed only a weak cytotoxic activity until 1 hour of incubation. Interestingly, during this period, A1 encapsulated formulations exhibited a moderate cytotoxic activity, the percentage of cell death reaching 30%, after 1 hour of incubation. During this period, since the inactive peptide A1Yala did not provoke any substantial cell death when encapsulated, we concluded that the activity of the archaeosome-A1 product was due to the activity of the A1 peptide that conserved its potential after encapsulation. We cannot formally exclude that this activity was due to the leakage of free peptide during storage or dilution, but we observed a similar kinetics of the encapsulated peptide after a 2 month storage period, and the archaeosomes were prepared in PBS, to avoid their perturbation when diluted in the culture medium RPMI that possesses a similar osmolarity, salt concentration and pH. Archaeosome cytotoxicity that appeared after one hour of incubation did not allow us to follow cell death kinetics of the encapsulated peptide, but during the first hour the effect of A1-archaeosome is only partial suggesting a slower dynamic for the encapsulated A1 compared with the non-encapsulated A1. Since the latter requires the penetratin sequence to be active,¹¹ in both cases peptide effects seem to be dependent of lipid membrane penetration. But the difference of dynamics suggests a specific mode of action of the A1 peptide when coupled to archaeosomes which could be explained by the required time to achieve endocytosis after interaction of the functionalized archaeosomes with folate receptors. These results are interesting for in vivo tests because archaeosomes are supposed to protect A1 peptides from degradation and facilitate their addressing to target cells.

Fig. 4 In vitro cytotoxicity of peptides A1 and AIYala encapsulated archaeosome formulations. HeLa cells were incubated with the indicated products or with the vehicle (PBS) control, in presence of 5 mM Syto13 and 12,5 μg mL⁻¹ propidium iodide. Cell death was calculated as the percentage of PI-positive and Syto-13 negative cells from images collected on a Leica SP5 fluorescent microscope. Archeo-A1 1.7 (archaeosomes made of 1.7 mg mL⁻¹ of lipids and containing 50 μM of peptide A1); archeo-A1 1.1 (archaeosomes made of 1.1 mg mL⁻¹ of lipids and containing 32.5 μM of peptide A1); archeo-A1YaLa 1.7 (archaeosomes made of 1.7 mg mL⁻¹ of lipids and containing 24.4 μM of peptide A1Yala); archeo-A1YaLa 1.1 (archaeosomes made of 1.1 mg mL⁻¹ of lipids and containing 15.5 μM of peptide A1Yala). For several points, error bars are too small to be seen.

Besides, it is important to note that free archaeosomes and peptide loaded archaeosomes expressed a similar cytotoxic activity after 3 hours of incubation. Indeed, after a certain period (>1 hour), the cytotoxicity of archaeosomes with lipid concentrations of 1.1 and 1.7 mg mL⁻¹ became significant, that may result from a ‘surfactant effect’ of the lipids inducing perturbations of the plasma membrane. Further in vitro and in vivo studies will be soon investigated to clearly explain the biological activity profile and to evaluate the potential of folate-equipped archaeosomes for the delivery of these original anticancer peptides.

3. Conclusions

In summary, we have developed a novel folate-targeted version of archaeosome technology suitable for the encapsulation of therapeutic peptides through the incorporation of limited amounts of new PEGylated archaeolipids into standard phospholipid-based vesicle formulations. The synthesis route for the preparation of the folate PEGylated tetraether was based upon efficient successive peptidic coupling reactions involving folic acid or carboxylic acid tetraether and PEGylated amine derivatives. Archaeosomes loaded with peptides were prepared by thin film hydration method followed by a lyophilization step of the liposomal dispersions and a rehydration process in an aqueous solution containing the valuable peptides. A convenient analytical LC-MS method was developed to determine encapsulation efficiencies. Preliminary in vitro evaluations of peptide-loading archaeosomes equipped with the folate ligand revealed an influence of the archaeosome formulations on peptide cytotoxicity especially during the first hour of incubation. Additional studies are now in progress to increase both encapsulation efficiencies and archaeosome concentrations with the aim of optimizing the therapeutic index of the drugs for in vivo applications.

4. Experimental

4.1. Materials and methods

Egg-PC was purchased from Sigma. MeO–PEG₂₀₀₀–tetraether–OH was synthesized according to a four-step procedure from the tetraether diol available in our laboratory.⁹ All reactions were carried out under nitrogen atmosphere with dry, freshly distilled solvents under anhydrous conditions. Dichloromethane (CH₂Cl₂) and methanol (MeOH) were distilled over calcium hydride. All other reagents were used directly from the supplier without further purification unless noted. Analytical thin-layer chromatography (TLC) was performed on Merck 60 F254 silica gel non-activated plates. A solution of 5% H₂SO₄ in EtOH or ultra violet fluorescence was used to develop the plates. Column chromatography was performed on silica gel MERCK 60 H (5–40 μm). Nuclear magnetic resonance spectra (¹H NMR and ¹³C NMR) were recorded on a Brucker ARX 400 instrument (¹H at 400 MHz, ¹³C at 100 MHz). Data are reported as follows: chemical shift (number of hydrogen, multiplicity, coupling constants if applicable). The chemical shifts (δ) are reported as parts per million (ppm) referenced to the appropriate residual solvent peak. Coupling constants are reported in Hertz (Hz). Abbreviations are as follows: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublet), m (multiplet).

4.2. Synthesis of FA–PEG₅₀₀₀–tetraether–OH 8

4.2.1. Synthesis of FA–PEG₅₀₀₀–NHBOC 3. Under nitrogen atmosphere, folic acid 1 (44 mg, 0.099 mmol; 5 equiv.), NHS (13 mg, 0.113 mmol, 5.5 equiv.) and DCC (41 mg, 0.198 mmol, 10 equiv.) were dissolved in 2.5 mL of dry DMSO and the mixture was stirred in the dark for 29 h at 25 °C. Then, it was filtered and a solution of BocNH–PEG₅₀₀₀–NH₂ 2 (100 mg, 0.02 mmol, 1 equiv.) in dry DMSO (4 mL) was added dropwise to the mixture and the reaction was stirred for an additional 48 h. The residue was purified by dialysis (Molecular Weight CutOff (MWCO) = 3500 Da) against DMSO to afford after lyophilisation a yellow powder (103 mg, 94%), as a 46 : 38 : 16 mixture of the two regioisomers γFA–PEG₅₀₀₀–NHBoc 3γ and αFA–PEG₅₀₀₀–NHBoc 3α and unreacted H₂N–PEG₅₀₀₀–NHBoc 2. δ_H (500 MHz; DMSO-d6) 1.37 (9H, s, H_BOC), 1.84 (0.27H, m), 1.89 (0.23H, m), 1.95 (0.27H, m), 2.01 (0.23H, m), 2.18 (0.45H, t, J 7.1), 2.24 (0.55H, t, J 7.7), 3.05 (2H, q, J 3.05), 3.47–3.69 (528H, m), 4.23 (0.45H, m), 4.35 (0.55H, m), 4.48 (2H, d, J 4.5), 6.63 (1H, d, J 8.9), 6.64 (1H, d, J 8.9), 6.73 (1H, t, J 5.9), 6.92 (1H, dd, J 5.7, 9.8), 7.63 (1H, d, J 8.9), 7.65 (1H, d, J 8.9), 7.85 (0.55H, t, J 5.7), 7.89 (0.45H, t, J 5.7), 7.97 (0.55H, d, J 7.9), 8.63 (0.55H, s), 8.64 (0.45H, s); δ_C (125 MHz; DMSO-d6) 27.41, 28.72, 31.43, 32.52, 38.95, 40.09, 52.51, 53.00, 64.22, 111.49, 122.02, 129.21, 149.12, 151.13, 155.88, 156.91, 166.63, 172.24, 174.66.

4.2.2. Synthesis of FA–PEG₅₀₀₀–NH₂ 4. The residue FA–PEG₅₀₀₀–NHBOC 3 (295 mg, 0.0542 mmol) was dissolved in 1 M HCl in HCO₂H (25.5 mL) and the reaction mixture was stirred for 4 h at room temperature in the dark. At 0 °C, an aqueous solution (1 M NaOH) was added until pH 10. The mixture was extracted with dichloromethane, and the resulting organic phase was washed with water, saturated NaHCO₃ and brine, dried (MgSO₄), and concentrated under reduced pressure. A yellow powder (282 mg, 97%) was obtained as a 46 : 38 : 16 mixture of the two regioisomers γFA–PEG₅₀₀₀–NH₂ 4γ and αFA–PEG₅₀₀₀–NH₂ 4α and H₂N–PEG₅₀₀₀–NH₂ 5. δ_H (500 MHz; DMSO-d6) 1.84 (0.27H, m), 1.89 (0.23H, m), 1.95 (0.27H, m), 2.01 (0.23H, m), 2.18 (0.45H, t, J 7.1), 2.24 (0.55H, t, J 7.7), 3.05 (2H, q, J 3.05), 3.47–3.69 (528H, m), 4.23 (0.45H, m), 4.35 (0.55H, m), 4.48 (2H, d, J 4.5), 6.63 (1H, d, J 8.9 Hz), 6.64 (1H, d, J 8.9), 6.92 (1H, dd, J 5.7, 9.8), 7.63 (1H, d, J 8.9), 7.65 (1H, d, J 8.9), 7.85 (0.55H, t, J 5.7), 7.89 (0.45H, t, J 5.7), 7.97 (0.55H, d, J 7.9), 8.63 (0.55H, s), 8.64 (0.45H, s); δ_C (125 MHz; DMSO-d6) 27.41, 31.43, 32.52, 38.95, 40.09, 52.51, 53.00, 64.22, 111.49, 122.02, 129.21, 149.12, 151.13, 155.88, 156.91, 166.63, 172.24, 174.66.

4.2.3. Synthesis of FA–PEG₅₀₀₀–tetraether–OAc 7. To a solution of HOOC–tetraether–OAc 6 (80 mg, 0.064 mmol) in dry dichloromethane (17 mL) were added NHS (8 mg, 0.0696 mmol), DCC (26 mg, 0.128 mmol) under nitrogen. The mixture was stirred for 24 h at 25 °C in the dark. The solution was concentrated under reduced pressure and to the residue were added dry DMSO (10 mL), pyridine (1 mL) and a solution of FA–PEG₅₀₀₀–NH₂ 4 (172 mg, 0.032 mmol) in dry DMSO (5 mL). The mixture was stirred for 4 days at 50 °C in the dark. The reaction mixture was purified by dialysis (molecular weight cutoff (MWCO) = 3500 D) against DMSO/CHCl₃ (1/1) to afford after lyophilisation a yellow powder (172 mg, 81 mol%), as a mixture comprising a 45 : 35 : 17 : 3 molar ratio of γFA–PEG₅₀₀₀–tetraether–OAc 7γ, αFA–PEG₅₀₀₀–tetraether–OAc 7α, FA–PEG₅₀₀₀–NH₂ 4 and H₂N–PEG₅₀₀₀–NH₂ 5. δ_H (400 MHz; DMSO-d6) 0.80–0.89 (31H, m), 1.02–1.81 (98H, m), 2.05 (3H, s), 3.16 (4H, d, J 6.9), 3.38–3.55 (544H, m), 3.96 (1H, dd J 6.6, 16.5), 4.03 (1H, dd, J 4.1, 11.6), 4.09 (1H, d, J 4.1, 10.5), 4.22 (1H, dd, J 4.1, 10.5), 4.38 (1H, m), 4.47 (2H, m), 6.63–6.83 (3H, m), 7.52–7.99 (5H, m), 8.57 (0.55H, s), 8.60 (0.45H, s). δ_C (125 MHz; CDCl₃/DMSO-d6; 80/20) 19.33, 19.23, 20.32, 21.17, 22.69, 24.32, 24.45, 24.77, 26.03, 26.07, 26.15, 27.93, 28.82, 29.32–29.84, 30.09, 30.64, 32.03, 32.84, 34.10, 36.83, 37.52, 37.61, 38.70, 39.27, 45.71, 53.65, 62.18, 67.53, 70.61, 70.94, 71.14, 71.85, 71.89, 75.88, 78.98, 112.28, 130.21, 149.06.

4.2.4. Synthesis of FA–PEG₅₀₀₀–tetraether–OH 8. To a solution of FA–PEG₅₀₀₀–tetraether–OAc 7 (165 mg, 0.025 mmol) in CHCl₃/MeOH (10 mL, v/v) was added 5 μL of MeONa in MeOH (30%, 0.027 mmol), and the reaction mixture was stirred at room temperature for 16 h. Amberlite resin (IR 120) was added, the reaction mixture was filtered, and the solvents were evaporated under reduced pressure. A yellow powder (171 mg, quant.) was obtained as a mixture comprising a 45 : 35 : 17 : 3 molar ratio of γFA–PEG₅₀₀₀–tetraether–OH 8γ, αFA–PEG₅₀₀₀–tetraether–OH 8α, FA–PEG₅₀₀₀–NH₂ 4 and H₂N–PEG₅₀₀₀–NH₂ 5. δ_H (400 MHz; DMSO-d6) 0.80–0.89 (31H, m), 1.02–1.81 (98H, m), 3.16 (4H, d, J 6.9), 3.38–3.55 (544H, m), 3.96 (1H, dd J 6.6, 16.5), 4.03 (1H, dd, J 4.1, 11.6), 4.09 (1H, d, J 4.1, 10.5), 4.22 (1H, dd, J 4.1, 10.5), 4.38 (1H, m), 4.47 (2H, m), 6.63–6.83 (3H, m),7.52–7.99 (5H, m), 8.57 (0.55H, s), 8.60 (0.45H, s). δ_C (125 MHz; CDCl₃/DMSO-d6; 80/20) 19.33, 19.23, 20.32, 22.69, 23.49, 24.51, 25.19, 25.76, 26.66, 26.07, 26.15, 27.78, 28.83–33.97, 34.05, 36.97, 37.04, 37.37, 37.46, 38.70, 39.41, 46.05, 53.53, 62.18, 67.53, 70.49, 70.94, 71.14, 71.85, 71.89, 74.90, 78.46, 111.82, 129.54, 148.93, 170.63, 171.59.

4.3. Archaeosome preparation and characterization

4.3.1. Preparation of folate equipped archaeosome formulations. Stock solutions of Egg-PC (24.3 mg mL⁻¹), MeO–PEG₂₀₀₀–tetraether–OH (9.9 mg mL⁻¹) 9 and FA–PEG₅₀₀₀–tetraether–OH 8 were prepared in MeOH. PEGylated archaeosomes were obtained by the hydration method as already described elsewhere.¹⁶ Briefly, the selected lipid solutions were mixed to yield a mixture of 90 wt% Egg-PC, 5 wt% of MeO–PEG₂₀₀₀–tetraether–OH 9 and 5 wt% of FA–PEG₅₀₀₀–tetraether–OH 8 with a total lipid concentration of 1 mg mL⁻¹. The organic solvents were then evaporated using a rotary evaporator and the lipid films thus obtained were dried under high vacuum for 5 hours at room temperature. The dried lipid films were then hydrated with 1 mL of milliQ water. The solutions were vortexed and left at 4 °C overnight. Thereafter, the suspensions were sonicated at room temperature for three times 5 min with intervals of 5 min using a Fischer Scientific sonication bath (FB 15051) at 80 kHz. Archaeosomal formulations were then freeze-dried in order to arrange dry pre-prepared vesicles for the encapsulation step.

4.3.2. Effect of DMSO on archaeosome size and polydispersity. Four freeze-dried archaeosomal formulations (1 mg each) were prepared using the above described method. Thereafter, they were respectively hydrated with 1 mL of milliQ water at 0%, 1%, 10% and 20% DMSO (molar ratios). The solutions were then sonicated at room temperature for 3 times or 7 times 5 min with intervals of 5 min using the same sonication bath described above. After a rest period of one hour, size and polydispersity index of mixtures were controlled. Each formulation was realized in duplicate.

4.3.3. Size and polydispersity measurements. The size (average diameter obtained by the cumulant result method) and the polydispersity index of the formulations were measured by dynamic light scattering using a Delsa™ Nano Beckman Coulter apparatus at 25 °C.

4.3.4. Cryo-TEM study. The cryo-TEM analysis of folate PEGylated archaeosomes was realized by Dr Olivier LAMBERT at the University of Bordeaux (Group “Chimie et Biologie des Membranes et Nano-objets”, UMR 5248 CNRS). Each sample (5 μL) was deposited on a grid covered with a carbon film having 2 μm diameter holes previously exposed to treatment with UV-ozone. The excess of water was removed by absorption with filter paper to form a thin layer of water suspended inside the holes. This grid was then plunged quickly (EM CPC, Leica) in liquid ethane (−178 °C). Rapid freezing of the thin layer of liquid water in vitreous ice (absence of crystals) preserved biological structures. Grids were then placed in a suitable object carrier for observing the samples at −170 °C. Observation under a microscope (FEI Tecna F20) was carried out in the mode low dose limiting the effects of beam irradiation on the lipid material. Images were recorded using an ultra-sensitive camera (Gatan, USC 1000) 2k × 2k with pixel size of 14 μm. The electron dose used was 10–20 electrons per Ų. The image resolution under these conditions was about 2 nm.

4.3.5. Peptide loading. Peptide encapsulation studies into PEGylated archaeosomal formulations were based on three steps in order to quantify the encapsulation efficiency. Firstly, HPLC separation conditions were promoted. Secondly, a LC-MS quantification method was developed. Thirdly, an encapsulation procedure was optimized.
4.3.5.A. Development of HPLC conditions. A LC-MS2020 from Schimadzu with an ESI detection was used to separate the peptide from lipid mixture (Egg-PC, 8 and 9). An analytical solution containing the desired peptide mixed to lipids in MeOH was injected in the apparatus which was already connected to an Accalaim Column 120 C18 120 A 2.1 × 100 mm. A mixture of H₂O (0.1% HCOOH) and ACN (0.1% HCOOH) was used to elute compounds. The best separation conditions were found using a gradient up to 100% ACN (0.1% HCOOH) at a flow rate of 0.4 mL min⁻¹. Each experiment was performed in duplicate.
4.3.5.B. Quantification method by LC-MS. A calibration curve was expanded for a peptide concentration ranging from 5 to 20 μM by linearization the area of the LC-MS chromatogram in function of the concentration of the sample. The correlation coefficients of all calibration curves were greater than 0.99. All experiments were performed in duplicate. And in order to ensure that the developed LC-MS analytical method is reproducible and accurate, the same sample of each peptide was injected 6 times to control the values of the measured area of the chromatogram (see ESI†). Also, we opted to realize the calibration curve experiments and to quantify the desired peptide on the same day.
4.3.5.C. Peptide encapsulation. 1 mg of freeze-dried pre-prepared vesicles obtained using the above procedure (3.3.1.) was re-hydrated with 1.0 mL of aqueous solution at 1% DMSO containing the desired amount of peptide (100 μM) and left at 4 °C for 5 hours. Archaeosome formulations were then sonicated at room temperature for three times 5 min with interval of 5 min. Unencapsulated peptide was removed using the ultrafiltration method. The samples were filtered through 50 000 NMWL pore filters (Amicon Ultra 2 mL 50 K, Ref: UFC 205024) by ultracentrifugation at 7500g for 1 hour at 15 °C. The determination of encapsulation efficiency was performed by recovering the supernatants and the filtrates with MeOH to disrupt the vesicle structure. The presence of peptide in each fraction was quantified by LC-MS using the new analytical method described above (4.3.5.B). The encapsulation efficiency was then calculated from the ratio between the concentration of peptide in the vesicle solution (pellet) and the initial concentration of peptide. Encapsulation efficiencies of 18% with peptide A1 and 9% with A1Yala were obtained especially with archaeosomes loaded (m_peptide/m_lipids) with 4.1 wt% and 1.9 wt% of peptide respectively.

4.3.6. Preparation of formulations for biological studies. With regard to biological assays, the same encapsulation procedure described above (4.3.5.C) was employed at higher quantity by splitting the desired lipid amount (32 mg) on 8 flasks in order to ensure the reproducibility of results. The hydration period of the lyophilized empty vesicles by the aqueous peptide solution was extended to 23 h instead of 5 h as described above in order to increase the encapsulation efficiency. After the sonication step (3 times 5 min with an interval of 5 min), the solution was submitted to ultracentrifugation at 7500 g for 1 h at 15 °C. After removal of the supernatant containing the non-encapsulated peptides, the remaining pellet was resuspended in 9.18 mL of PBS buffer for A1 formulation (100 μM, encapsulation efficiency 30%) and in 4.84 mL for A1Yala formulation (100 μM, encapsulation efficiency 14%). For each experiment, the concentration of the peptide was controlled in the filtrate and the pellet fractions. Values obtained were very close to that of the initial concentration of the peptide. In the same order, 6.86 mL of empty vesicles were prepared in PBS buffer at a total lipid concentration of 11 mg mL⁻¹ with regard to negative biological controls.

4.4. Evaluation of in vitro antitumor activity

4.4.1. Cell culture. HeLa cells were grown in complete Dulbecco's modified Eagle medium (Gibco BRL) supplemented with 10% heat-inactivated fetal calf serum (FCS),1 unit per mL penicillin, 1 μg mL⁻¹ streptomycin, 1% L-glutamine, (Gibco BRL), at 37 °C in a 5% CO₂ humidified atmosphere.

4.4.2. Time-lapse microscopy. 10 000 cells per well were plated in 96 well plates with glass bottoms in RPMI 1640 (Gibco BRL, Life Technologies) supplemented with 1 unit per mL penicillin, 1 μg mL⁻¹ streptomycin, 1% L-glutamine, (Gibco BRL) and grown overnight. Before adding peptide or archaeosomal formulations, 5 μM Syto 13 (Molecular Probes, Gibco BRL) and 12,5 μg mL⁻¹ propidium iodide (PI) (Sigma) were added to the culture medium.

Fluorescence was followed at various time intervals with a Zeiss Observer Z1 microscope (Zeiss, Marly Le Roi, France).

Acknowledgements

This work was financially supported by the Region Bretagne (grant to AJ). We are grateful to Jean-Paul Guégan for help with NMR studies and Jocelyne Le Seyec (ID2Santé) for assistance during the project.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: ESI-MS spectra and HPLC chromatograms of peptides A1 and A1Yala and additional data relative to the quantitative determination of peptide encapsulation efficiency. See DOI: 10.1039/c6ra15713k

‡ Present address: Univ Paris Diderot, Sorbonne Paris Cité, Epigenetics and Cell Fate, UMR 7216, CNRS, Paris F-75013 (France).

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