Biodegradable and Dual‐Responsive Polypeptide‐Shelled Cyclodextrin‐Containers for Intracellular Delivery of Membrane‐Impermeable Cargo

Abstract The transport of membrane impermeable compounds into cells is a prerequisite for the efficient cellular delivery of hydrophilic and amphiphilic compounds and drugs. Transport into the cell's cytosolic compartment should ideally be controllable and it should involve biologically compatible and degradable vehicles. Addressing these challenges, nanocontainers based on cyclodextrin amphiphiles that are stabilized by a biodegradable peptide shell are developed and their potential to deliver fluorescently labeled cargo into human cells is analyzed. Host–guest mediated self‐assembly of a thiol‐containing short peptide or a cystamine‐cross‐linked polypeptide shell on cyclodextrin vesicles produce short peptide‐shelled (SPSVss) or polypeptide‐shelled vesicles (PPSVss), respectively, with redox‐responsive and biodegradable features. Whereas SPSVss are permeable and less stable, PPSVss effectively encapsulate cargo and show a strictly regulated release of membrane impermeable cargo triggered by either reducing conditions or peptidase treatment. Live cell experiments reveal that the novel PPSVSS are readily internalized by primary human endothelial cells (human umbilical vein endothelial cells) and cervical cancer cells and that the reductive microenvironment of the cells’ endosomes trigger release of the hydrophilic cargo into the cytosol. Thus, PPSVSS represent a highly efficient, biodegradable, and tunable system for overcoming the plasma membrane as a natural barrier for membrane‐impermeable cargo.


General Procedures
Preparation of cyclodextrin vesicles (CDV): Unilamellar bilayer vesicles of amphiphilic cyclodextrin derivatives were prepared by hydration of a thin film and subsequent extrusion. The solvent was evaporated in a stream of argon to obtain a thin film and residual solvent was removed under high vacuum. The film was hydrated by addition of HEPES to yield a total amphiphile concentration of 100 µM. For loaded containers CDVcargo, pyranine (5 mM), phalloidin488 (20 µM) or α-amanitin (0.3 mM) solution in HEPES was added and the amphiphilic cyclodextrin film was hydrated. After vigorous stirring overnight, this solution was vortexed, and repeatedly passed through a polycarbonate membrane with 100 nm pore size (AVESTIN) in a Liposofast manual extruder (AVESTIN) to yield CDV.

Preparation of Short-peptide shelled vesicles (SPSVSS):
For the case of Ad-GGCCDD, Ad-TEG-GGCCDD or Ad-GGGCCCDDD, 25 µL of 2 mM Ad-GGCCDD or Ad-TEG-GGCCDD or Ad-GGGCCCDDD in HEPES buffer was added to 1 mL 100 µM CDV and stirred for 15 min to give SPSVSH. After stirring in an open vial for aerial oxidation overnight SPSVSS were obtained. For the case of Ad-GGGDDDD, 25 µL of 2 mM Ad-GGGDDDD in HEPES buffer was added to 1 mL CDV and stirred for 15 min to give SPSVCOOH. 1.6 µL of 1 M EDC.HCl in HEPES buffer was added to SPSVCOOH and stirred for 30 min followed by 2 µL of 50 mM of cystamine in HEPES buffer and stirred overnight to obtain SPSVSS.
Preparation of polypeptide shelled vesicles (PPSVSS): 25 µL of 2 mM Ad-PLG105 in HEPES buffer was added to 1 mL CDV and stirred for 30 min to yield PPSVCOOH. 50 µL of 1 M EDC•HCl in HEPES buffer was added to PPSVCOOH and stirred for 30 min followed by 25 µL of 50 mM of cystamine in HEPES buffer and stirred overnight. Non-encapsulated dyes were separated by gel filtration.
Preparation of Dy633 conjugated PPSVSS: 25 µL of 2mM Ad-PLG105 in HEPES buffer was added to 1 mL 100 µM CDV and stirred for 30 min to yield PPSVCOOH. 50 µL of 1 M EDC•HCl in HEPES buffer was added to PPSVCOOH and stirred for 30 min followed by addition of 25 µL of 50 mM of cystamine in HEPES buffer and stirred overnight. 20 µL of 5 mM Dy633 was added to this solution and stirred for 6 h. Non-conjugated dye and other reagents were removed by dialysis against HEPES buffer.
Uptake: For analyzing the time dependent uptake, freshly prepared PPSVSS were dialyzed against M199, diluted 1:4 in serum free high glucose DMEM and added to HUVEC for the times indicated in a live cell experimental setup (37 °C and 5% CO2). For all other uptake experiments, the freshly dialyzed PPSVSS were diluted in M199 and incubated with a HUVEC cell layer for 30 min at 37°C and 5% CO2. After washing 5 times with M199, cell growth was continued in a 1:1 mixture of ECGM2 and M199 with 10% FCS. For uptake of PPSVSS in HeLa cells same procedure was used as described but the incubation setup was changed to 37 °C and 7% CO2.
Costaining with endosome marker: For colocalization experiments, FITC dextran 10 kDa (Thermo Fisher Scientific) (dissolved in DMSO to 1 mM stock solution) was used. The marker was diluted to a 1 µM concentration and applied to the cells together with the PPSVSS for 30 min.
Lactate dehydrogenase (LDH) assay: Cytotoxicity of PPSVSS on HeLa cells or HUVEC was assessed by measuring the lactate dehydrogenase (LDH) activity released from damaged cells. The cells were seeded in 96-well plates with a density of 3000 cells/well and cultured for 1 d. Subsequently, the culture medium was replaced with assay medium containing different PPSVSS concentration, ranging from 0 to 50 µM in serum-free medium. The plates were left in the incubator either for 2 h or 4 h at 37°C. The maximal LDH activity that represents the control values was determined by addition of 1% Triton X-100 for 10 min to completely lyse the cells. After transfer of the cell supernatant to a new 96-well plate, they were mixed in a volume ratio of 1:1 with reaction mixtures containing the tetrazolium salt, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5phenyl-2H-tetrazolium (INT), followed by incubation for exact 30 min at 37°C, protected from light. The reaction was stopped by addition of 50 µL HCl (1 N) to each well. The values of relative toxicity (percentage of LDH activity as compared to the maximal value obtained after Triton treatment) were calculated after measuring the absorbance at 492 nm on a Microplate Reader. The concentration-response curve obtained was fitted to a growth-sigmoidal function. LDH assays were carried out for each sample in triplicate (n = 3).
Flow cytometric analysis of PPSVSS,pyr internalization: Cells were treated with PPSVSS,pyr for for the given amount of time at 37 °C. Cells were then washed in PBS (Sigma), and detached in accutase solution (Sigma) for 3 min at 37 °C. 7AAD (eBioscience, San Diego, CA, USA) allowed the exclusion of dead cells. A Guava easyCyte™ System (Millipore, Burlington, MA, USA) was used to determine the percentage of pyranine positive (excitation at 450 nm) cells per 10,000 cells. PPSVSS were used as a negative control. FACS assays were carried out for each sample in triplicate (n = 3).

Cell viability assay:
The cytotoxicity of PPSVSS, amanitin was assessed in HeLa cells and HUVEC. Cells (2,000 cells/well) were added to the wells of a 96-well plate (Corning, Woburn, MA, USA). After culturing at 37 °C for 24 h, the cells were incubated with PPSVSS, amantin at either 0, 10, 20, 30, 40 or 50 µM (in M199 Medium) for 4h, washed and then cultivated for further 24 h. The proportion of viable cells was evaluated using a CCK-8 kit (Sigma-Aldrich, St. Louis, MO,catalog No. 96992) or phase contrast microscopy to count adherent viable cells after treatment of PPSVSS, amanitin. Blank wells only with culture media and PBS-treated wells were used to define 0 and 100% viability, respectively. CCK-8 assays were carried out for each sample in triplicate (n = 3).

Chemical Synthesis
General: All reactions were carried out in heat-gun-dried glassware under argon atmosphere and were performed by using standard Schlenk techniques. Thin layer chromatography was carried out on Merck silica gel 60 F254 plates; detection by UV or dipping into a solution of KMnO4 (1.5 g), NaHCO3 (5.0 g) in H2O (400 mL) followed by heating. Flash chromatography (FC) was carried out on Merck silica gel 60 (40 -63 μm) at an argon pressure of 0-0.5 bar.

Short peptide synthesis
Synthesis of Ad-GGCCDD: Peptides were synthesized using solid phase peptide synthesis (SPPS).
DIPEA (2 eq) was added and the mixture was agitated for 5 min by the argon stream. A second portion of DIPEA (3 eq) was added and then agitated for 2 h by the argon stream. Methanol (1 ml/g resin) was added and the resulting mixture was agitated for 15 min to quench the remaining resin functionalities. After filtration of the reaction mixture the resin was washed with DCM (3 x 30 ml), DMF (3 x 30 ml), DCM (3 x 30 ml) and methanol (3 x 30 ml). The resin was dried under vacuum. The elongation was performed using an automated peptide synthesizer. After transferring the dry resin to the reaction vessel, it was pre swollen by shaking in DMF (20 ml) for 5 min and then washed with DMF (2 x 20 ml) The Fmoc protecting group was cleaved by shaking in 20% piperidine solution in DMF (20 ml). After removing the solution another portion of 20% piperidine solution in DMF (20 ml) was added and shaken for 20 min to ensure complete deprotection. The resin was washed with DMF (7 x 20 ml) and the second Fmoc-protected amino-acid (Fmoc-ASP(OtBu)-OH) (3 eq. relative to resin loading, 0.5 M solution in DMF) was added. HOBt (4 eq, 0.4 M solution in DMF) and DIPCDI (4 eq, 0.4 M solution in DMF) were added and the mixture was shaken for 2.5 h. After washing with DMF (3 x 20 ml) the procedure was repeated for Fmoc-Cys(Trt)-OH two times, Fmoc-Gly-OH two times and with adamantane acid. After the completion of the addition of amino acids, the resin was suspended in a solution of TFA:H2O:Triisopropylsilane:edt (95:2.5:2.5:1, 20 ml) and stirred overnight to cleave from the resin and to remove the protecting groups. The reaction mixture was sucked off and the resin was washed with TFA and peptides then precipitated by the addition of cold Et2O. After collecting the precipitate by centrifugation, it was further purified by using RP-MPLC with the eluents 0.05% TFA in water and acetonitrile. Other peptides Ad-TEG-GGCCDD, Ad-GGGCCCDDD, Ad-GGGDDDD were synthesized accordingly. Product formation was confirmed by MALDI TOF or HRMS and the purity was also confirmed by RP-MPLC. (1) 2-(2-(2-chloroethoxy)ethoxy)ethan-1-ol (25 g, 148.26 mmol) in 50 ml dry THF was mixed with a catalytic amount of sodium under argon atmosphere and stirred until it dissolved completely. Tertbutyl acrylate (24.7 g, 192.74 mmol) was added and stirred for 24 h at room temperature. After completion of the reaction, monitored by TLC, 10 mL of 1 M HCl was added to the reaction mixture and stirred for 10 minutes. THF was removed under reduced pressure and the rest of the solution was suspended in 100 mL brine solution for 15 minutes. The product was extracted to ethyl acetate (3 x 80 mL) and pure product was obtained as a colourless liquid after column chromatography using ethyl acetate and cyclohexane as an eluent. (yield = 80%). 1 H NMR (400 MHz, CDCl3, δ): 3.62 (m, 14H, CH2O), 2.46 (t, 2H, CH2CO), 1.4 (s, 9H, CH3); 13 C NMR (100 MHz, CDCl3, δ): 170.59, 80.18, 71.07, 70.38, 70.32, 70.29, 70.10, 66.62, 42.42, 35.99, 27.81 HRMS (ESI) m/z: [M + Na] + calcd for C13H25ClO5, 319.1391; found, 319.1282.