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Keywords:

  • α1-antitrypsin;
  • cell-penetrating peptides;
  • chronic inflammation;
  • drug delivery;
  • human leukocyte elastase

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. LITERATURE CITED

α1-Antitrypsin (AT), a serine protease inhibitor that specifically targets hydrolytic enzymes, plays a significant role in the termination of tissue inflammation and can therefore represent a key factor in chronic incidences as chronic obstructive pulmonary disease (COPD) or chronic hepatitis. A local and low-dose therapy for the treatment of acquired chronic inflammatory processes which are characterized by insufficient AT amounts but also of genetically conditioned AT deficiencies is supposed to be more effective and less cost-intensive compared to current therapies. In this study, a noncovalent complex formation between the cell-penetrating peptide carrier hCT(18-32)-k7 and AT was performed. The complex was applied to HEK293T/17 cells, as proof-of-principle, and polymorphonuclear leukocytes (PMN), which are responsible for tissue destruction and the perpetuation of inflammation in chronic processes. Both cell species show a successful uptake and subsequently both, an intracellular dot-shaped and homogeneous distribution of the complex demonstrating phagolysosomal as well as cytoplasmic availability. Furthermore, a decreased human leukocytic elastase (HLE) activity was observed after the direct complex administration to PMN. Since the application did not cause an enhanced vitality loss, the complex could facilitate an improvement in direct, local and low-dose treatment of chronically proceeding processes in order to attenuate protease-mediated tissue destruction. © 2013 International Society for Advancement of Cytometry

Several diseases of civilization are accompanied by chronic inflammation, which at least partly cause severe complications as tissue destruction and pain (1).

For instance, human leukocytic elastase (HLE) is characterized by a highly proteolytic specifity and a significant potential to cause tissue destruction, e.g. the acute lung damage in adult respiratory distress syndrome as well as the chronic development of emphysema (2, 3). Its natural HLE antagonist, α1-antitrypsin (AT), is a 52-kDa serine protease inhibitor (serpin) with a pI between 4.9 and 5.1 (4), which inactivates HLE and forms enzyme/inhibitor complexes during the infiltration of inflamed tissues by activated polymorphonuclear leukocytes (PMN). In patients with inflammatory arthropathies, significantly higher levels of inactivated AT in their synovial fluid well-correlating with elevated elastase-levels could be found (5). The local balance impairment between proteases and their naturally occurring inhibitors at inflamed tissue sites suggests a critical role of AT regarding tissue damage limitation and termination of chronic processes. Although AT inhibits HLE at physiological concentrations, higher AT concentrations especially during chronic processes are required since its inhibitory activity is additionally reduced by PMN-released reactive oxygen species (ROS) reacting with methionine-residues at its active site (6).

Current treatment methods are rather unsatisfying, cost-intensive and cause severe side-effects. Although augmentation therapy that is applied in the case of AT deficiency is well-tolerated, life-long weekly infusions are not well accepted by patients (7). Furthermore, immunosuppressants, glucocorticoids or nonsteroidal antirheumatic agents used for the treatment of chronic incidences have numerous drawbacks; they induce immune system impairment and increase the risk of infections as well as cardiovascular toxicity. Hence, there is a high demand for a gentle and side-effectless medication (8–10). Marginal delivery capacity as well as low availability of some new potent therapeutic molecules, as peptides, proteins, or nucleic acid therapeutics, restricts their application in a conventional way. In addition, a low in-vivo stability, the lack of cellular uptake and an inefficient capability to reach targets is associated with a complete loss of pharmaceutical potency and mostly requires high doses increasing the risks of major side effects (11).

Therefore, the direct cellular administration of active agents performed by means of an efficient drug delivery system could be the basis for new and elegant treatment approaches in a modern world. The advantage of a locally applied drug carrier based medication avoids negative effects which may occur during systemic drug usage. Delivery technologies with the advantages to improve cellular uptake of therapeutic molecules, to provide drug activity at low doses and low cytotoxicity can be regarded as a milestone in therapeutic development (11). Beside a multitude of carrier systems based on viral systems, nano- and microcapsules, -particles and -spheres, another promising vector system, the so-called cell-penetrating peptides (CPPs) are getting more interest for the translocation of covalently and noncovalently linked large and polar cargos. CPPs constitute a very promising tool for the noninvasive cellular import of cargos as small molecules, nucleic acids, peptides, proteins, liposomes, and particles (11). Recently, CPPs based on human calcitonin (hCT) have been developed and demonstrated to be efficient tools for drug delivery applications. HCT itself is a native peptide hormone composed of 32 amino acids, which is physiologically secreted by the thyroid gland and involved in the calcium regulation metabolism. HCT-derived peptides possess cell-penetrating properties and can be used for the transport of various cargos (12). The synthesis of branched hCT-derived peptides with a high number of basic residues caused advanced carrier function. By using these CPPs transfection of several cell lines, including primary cells, was demonstrated (13).

The association of AT deficiency with several chronic incidences as chronic obstructive pulmonary disease (COPD) or chronic hepatitis requires its application for the re-adjustment of the protease/antiprotease balance and subsequently for homeostasis restoration. Until now, augmentation therapy is the only available, well-tolerated and specific therapy for AT deficiency. However, a local complex injection allowing CPP-mediated AT-administration into immuno-reactive cells and tissues would offer a new and elegant pharmacological approach for the treatment of tissue injury; e.g. as a therapy for AT deficiency (14). Moreover, a similar approach could be effective for the treatment of chronic proceeding incidences.

The aim of this study is the efficient transport of functional AT to target cells by means of complex formation between CPPs and AT protein. For this purpose, we investigated AT/CPP complexes with respect to their cellular uptake and pharmacological activity. We found that direct administration of complexed AT to PMN allows HLE inhibition at the place of its origin, within cell compartments, and subsequently avoids HLE release into tissues.

With this targeted approach a low-dose therapy might be allowed, which is side-effectless, gentle and patient-friendly facilitating AT availability as well as the termination of several chronic processes.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. LITERATURE CITED

Protein Labeling

Protein labeling was performed according to Pierce® fluorescein isothiocyanate (FITC)/rhodamine isothiocyanate (RITC) antibody labeling Kits (Thermo Fischer Scientific, Bonn, Germany) with slight modifications. 2 mg AT (Sigma-Aldrich, Taufkirchen, Germany) have been dissolved in 500 μl borate buffer (50 mM sodium borate, pH 8.5) to give a final concentration of 4 mg/ml. Prepared protein solution was mixed with FITC/RITC reagent and incubated for 60 min at room temperature. Removal of excess FITC/RITC was performed by gel filtration. By measuring the absorbance at 280 nm and 495 nm (Amax of FITC) FITC/protein ratio could be determined as 0.25. By measuring the absorbance at 280 nm and 556 nm (Amax of RITC) RITC/protein ratio could be determined as 0.466.

Peptide Synthesis and Labeling

The cell-penetrating peptide hCT(18-32)-k7 (CPP) as well as its carboxy fluorescein-labeled analog CPP-CF were synthesized by automated peptide synthesis on a multiple Syro II peptide synthesizer (MultiSynTech, Witten, Germany) followed by the Fmoc/tBu-strategy utilizing a double coupling procedure and in-situ activation with HOBt/DIC as previously or peptide sequence and synthesis details refer to (13). The peptides were prepared as C-terminal amide using Rink amide resin as solid support.

The peptides were cleaved from the resin using trifluoroacetic acid (TFA/thioanisole/p-thiocresole (90:5:5 (v/v/v)) for 3 h at room temperature, and precipitated by addition of ice-cold diethyl ether. The peptides were purified and analyzed according to (13).

Complexation

1.98 μl (4.17 mmol/l) of labelled or unlabelled CPP (4 mg/ml, in aqua dest.) and 135 μl (8.3 μmol/l) of labeled or unlabeled AT (2 mg/ml, in aqua dest.) were mixed with 463.02 μl PBS and incubated for 30 min at 37°C. Therefore, the AT/CPP molecular ratio can be regarded as 1:500. To prove protein/peptide complex formation gel filtration was performed.

Peptide- and protein-labeling were adjusted to the following experiments. Control experiments with peptide only contained CPP-CF (data not shown). For investigations of complex/cell interactions by flow cytometry (FCM) AT-FITC/CPP and by confocal laser scanning microscopy (CLSM) AT-RITC/CPP were used. Ingestion of the AT-loaded CPP by HEK293T/17 cells was determined by trypanblue quenching (0.08%, 30 min, 4°C).

Gel Filtration

Gel filtration was performed by a PD10-column (exclusion size 5000 Da, 8.5 ml bed volume, desalting columns, GE Healthcare, Munich, Germany). Columns were equilibrated with 25 ml PBS. 600 μl of samples (0.27 mg AT alone and after a second washing/equilibration step the complex mixture containing AT/CPP-CF) were applied to the top of the column bed. After complete entering of the sample into the column bed 1.9 ml PBS were added. Eluate was discarded and the column was filled with another 7.5 ml PBS. 6.5 ml of eluate was collected (0.5 ml per vial). Column was equilibrated with 25 ml PBS and stored at 4°C. Although, the column can only distinguish between free peptide and molecules/complexes of molecular mass above 5000 Da, while both, free protein and CPP-complex pass the column without any retardation, complex formation verification is possible by means of fluorescence measurements. Complexed AT/CPP-CF will be fluorescence-positive while free AT will be fluorescence-negative.

Bradford Protein Determination

Bradford solution (Sigma-Aldrich, Taufkirchen, Germany) was filtered to remove blue floating particles. 500 μl eluate samples were incubated with 2.75 ml Bradford-reagent for 5 min. Absorbance was measured at 595 nm.

Fluorescence Spectroscopy

For the correlation of peptide/protein binding CPP-CF fluorescence was measured by means of fluorescence spectroscopy (Fluoro-Max-2, Horiba Jobin Yvon, Bensheim, Germany). Excitation was performed at 488 nm and emission was measured between 480 and 640 nm. The highest peak at 521 nm was correlated against the eluation volume.

Cell Culture and PMN Isolation

Human embryonic kidney (HEK 293T/17) cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). DMEM with L-glutamine was used as cell culture medium (PAA laboratories) added with 10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich, Taufkirchen, Germany) and 100 U/ml penicillin/100μg/ml streptomycin (Sigma-Aldrich, Taufkirchen, Germany). Cells were cultivated in a humidified atmosphere of 5% CO2 at 37°C.

PMN were isolated from human whole blood from healthy volunteers after informed signed consent was obtained as described before (15).

Complex Mixture/Cell Interaction

Cells were seeded in eight-chamber slides which were purchased from Nalge Nunc International (Napperville, IL; size of one well: 0.7 cm × 0.7 cm; each well contained 5 × 105 HEK 293T/17 cells, 2.5 × 104 U937 cells or 5 × 104 PMN).

After 24 h of growth in culture medium the HEK 293T/17 cells were incubated with the peptide, protein or the peptide/protein complex mixture (250 μl) in reduced culture medium (Optimem, GIBCO, Invitrogen, Darmstadt, Germany, 350 μl) for 1 h at 37°C. Vitality experiments were performed at 1 h as well as at 6 h.

PMN were isolated from human whole blood and seeded (5 × 104 cells) inside the chamber slights. After 1 h PMN became adherent and co-incubation experiments were started. The peptide, protein and peptide/protein complex mixture were incubated for 1 h as described before. Co-localization experiments related to the endo-/phagosomal uptake and concomitantly using the lysosomal staining Lysotracker Green were performed for 3 h. Vitality experiments were performed at 1 h as well as at 6 h.

Afterwards, all cells were washed three times with PBS and analyzed as described in the specific experimental section. HEK 293T/17 cells were trypsinized, centrifuged, resuspended in PBS, and immediately analyzed by flow cytometry. Cells used for CLSM were analyzed at the cover slides without trypsination. PMN could be removed from the chamber slides by slight shear forces. PMN (3 × 105) used for AT activity measurement after co-incubation were placed into eppendorf tubes and incubated with 250 μl peptide, protein or complex mixture for 1 h at 37°C. After two washing steps (PBS, 400g, 3 min) cells were resuspended in Hanks balanced salt solution (HBSS) + Ca2+ (60 μl) and stimulated with 0.1 μmol/l PMA for 30 min at 37°C. PMN were centrifuged (3000g, 5 min) and supernatant was used for activity measurements. Control samples containing supernatant and peptide, protein or complex mixture were additionally performed.

AT Effects on Elastase Activity

The supernatant of stimulated PMN was added to 10 μl elastase substrate (MeOSucc-Ala-Ala-Pro-Val-p-nitroanilide, Calbiochem, Darmstadt, 1 mmol/l, in Tris 200 mmol/l, pH 8) and filled with PBS for a final volume of 100 μl. The influence of pure AT, pure peptide and the complex mixture added to the supernatant was investigated. Another control was performed by the addition of pure AT as well as CPP to PMN before stimulation. PMN containing complex mixtures, AT or CPP were washed three times in PBS after 1 h incubation. HLE activity was measured at 410 nm (Infinite M200, Tecan, Männedorf, Switzerland) in a 96-well plate.

Vitality Assay—Detection of Annexin V-FITC—Binding to Phosphatidylserine and Propidium Iodide Intercalation into DNA

Binding of annexin V to phosphatidylserine (PS) was used to detect apoptotic cells after AT/CPP influence. Cell samples were centrifuged (400g, 5 min) and resuspended in binding buffer (ApoAlert® Annexin V-FITC apoptosis kit, Clontech Lab, Mountain View, CA). After addition of 5 μl FITC-conjugated annexin V solution, samples were incubated in the dark at room temperature (16). Propidium iodide (PI; 10 μl), a DNA-intercalating agent, was added to determine the vitality state of the cells. To generate non-vital cells as positive control, HEK293/17 cells and PMN were incubated with staurosporine (Sigma-Aldrich, Taufkirchen, Germany, 5 μg/ml) for 1 as well as for 6 h at 37°C. The samples were used for flow cytometric analyses after washing and resuspending in PBS.

Choleratoxin B Staining

Twenty-five microliter Choleratoxin B (CTB)-FITC (Sigma-Aldrich, Taufkirchen, Germany, 5 mg/ml, 1:100, in PBS) were incubated with HEK 293T/17 in eight-well chamber slides for 1 h at room temperature. After incubation the wells were washed three times with PBS and analyzed by CLSM.

Lysosomal Staining

Lysosomes were stained with Lysotracker Green DND-26 (50 nmol/l, Invitrogen, Karlsruhe, Germany) for 1 h at 37°C. After incubation the chambers were washed three times with Optimem and analyzed by CLSM.

Flow Cytometry

The fluorescence intensity of the cells was detected by FCM (FACSCalibur, Becton Dickinson, USA) with a laser excitation wavelength of 488 nm (FITC/CF detection in channel FL1, RITC/PI detection in FL2). FITC-fluorescence measurable in fluorescence channel 2 was compensated. 104 events in the cell region R1 of each measurement were analyzed using the WinMDI 2.9 software.

Confocal Laser Scanning Microscopy

Confocal microscopy images were recorded with a Zeiss LSM 510 Meta Laser Scanning Microscope equipped with a Plan-Apochromat 63×/1.4 oil objective (Zeiss, LSM 510 META, Jena, Germany). For the FITC-/Lysotracker Green fluorescence an Ar/Kr laser (excitation wavelength: 488 nm) was used, while RITC fluorescence was detected with a He/Ne laser (excitation wavelength: 543 nm). FITC- and Lysotracker Green emission were detected with band pass filter (505–525 nm and 475–525 nm), whereas RITC emission was detected by means of a long pass filter (LP 560 nm).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. LITERATURE CITED

First we examined the complex formation between AT and fluorescently labeled hCT(18-32)-k7 by co-incubation of protein and peptide in a 1:500 ratio. The chosen protein/peptide ratio of 1:500 is related to studies describing an efficient uptake not below ratios of 1:320 after an incubation period of 1 h (17). In our studies we examined different protein/peptide ratios from 1:50, 1:200 to 1:500 but we found the latter one to be the optimal ratio. Then the complexes were separated from unbound peptide by gel filtration and the successful complex formation was verified by fluorescence spectroscopy (Fig. 1). Furthermore, we determined the protein content (Fig. 1) in each fraction by Bradford assay. Although, the column can only distinguish between free peptide and molecules/complexes of molecular mass above 5,000 Da, while both, free protein and CPP-complex pass the column without any retardation, complex formation verification is possible by means of fluorescence measurements. Complexed AT/CPP-CF will be fluorescence-positive while free AT will be fluorescence-negative. The elution profiles for free AT follows the same pattern as the shown protein concentration in the complex. Because of the significant size difference of AT (52 kDa) and peptide (3 kDa) and the subsequent separation of both molecules, elution profiles for CPP alone are not shown. The coexistence of the protein band and a high CPP-fluorescence intensity confirms a successful complex formation between AT and hCT(18-32)-k7.

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Figure 1. The complex formation between AT and hCT(18-32)-k7. Shown is the formation of a stable complex of AT and hCT(18-32)-k7 which could be experimentally verified. The protein (8.3 μmol/l) and the CF-labeled peptide (4.17 mmol/l) were co-incubated (molar ratio 1:500) and analyzed by gel filtration at a PD10 column. Sample volumes of 0.5 ml were collected. The protein content was determined by Bradford protein assay (black squares). The fluorescence intensity (a.u.) of the CF-labelled CPP was analyzed by fluorescence spectrometry at 521 nm (striped bars). Although, the column can only distinguish between free peptide and molecules/complexes of molecular mass above 5,000 Da, while both, free protein and CPP-complex pass the column without any retardation, complex formation verification is possible by means of fluorescence measurements. Complexed AT/CPP-CF will be fluorescence-positive while free AT will be fluorescence-negative. Representative results of three independent experiments are shown.

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The next experiments are illustrating the successful AT delivery into cells. In a first approach we investigated HEK293T/17 cells as a proof-of-principle cell system. Therefore, cells were co-incubated with the AT/CPP complex for 1 h. Instead of unlabeled AT, FITC or RITC-labeled AT was used allowing intracellular protein localization (Fig. 2). The HEK 293T/17 cell population is gated according R1 (panel a). After co-incubation of the complex and HEK293T/17 cells the fluorescence intensity of the cells increased by a factor of 2.5 compared to autofluorescent cells (panel b, black curve, geometric mean (gmean): 4.8, untreated HEK293T/17 are shown as gray curve). Applying AT-FITC without hCT(18-32)-k7 as a control resulted in only a low fluorescence intensity increase (dark gray curve, gmean: 3.5) suggesting that AT alone is also capable to interact with HEK293T/17 cells but in a lower extent than the AT/CPP complex.

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Figure 2. HEK293T/17 cell–hCT(18-32)-k7 interactions. Shown is the adsorption and uptake of the AT-F/RITC/hCT(18-32)-k7 complex by HEK293T/17 cells measured by means of flow cytometry (a, b) and CLSM (ch). HEK293T/17 cells are gated according R1 (a) and used for further investigations. HEK293T/17 cells (5 × 105) were co-incubated with the stable protein-peptide complex (hCT(18-32)-k7, 4.17 mmol/l and AT-FITC, 8.3 μmol/l). The FITC-fluorescence intensity of HEK293T/17 cells is demonstrated in (b) (black). As control samples the FITC-fluorescence intensity of untreated (light gray) and HEK293T/17 cells incubated with AT-FITC (8.3 μmol/l, dark gray) is also demonstrated (b). A representative histogram of three independent experiments is shown. The binding of the complex was additionally proved by means of CLSM. HEK293T/17 cells (5 × 105) were co-incubated with the stable protein-peptide complex (hCT(18-32)-k7, 4.17 mmol/l and AT-RITC, 8.3 μmol/l). HEK293T/17 cells were counterstained with CTB. Cells treated with AT-RITC alone operating as control and their transmission are shown in panel (c) and (d). The RITC-fluorescence intensity of treated HEK293T/17 cells, suggesting adsorption and uptake of the AT/CPP complex, is demonstrated in panel (e). The transmission and CTB-counterstaining are shown in panels (f) and (g). The overlay is demonstrated in panel (h). Representative results of three independent experiments are shown. [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com.]

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Since FCM investigations are not revealing the nature of AT/CPP complex interaction with cells, such as providing information about membrane binding or internalization, the interaction of the AT/CPP complex with HEK293T/17 cells was also investigated by CLSM studies (Fig. 2, panels c–h). Therefore, a complex of unlabeled hCT(18-32)-k7 and RITC-labeled AT (panel h) compared to AT-RITC alone (panel c) was co-incubated with HEK293T/17 cells. In the latter case, we observed no intracellular fluorescence indicating that AT alone is not able to translocate into HEK293T/17 cells. To show the presence of a confluent cell layer, transmission image were added in panel d.

In contrast to the control, the complex can be localized within the cells and at cell membranes (panel e). Again, transmission image (panel f) shows the confluent cell layer. Interestingly, the applied complex seems to be both, enclosed in vesicles and distributed in the cytoplasm which is demonstrated by the dot-like as well as homogeneously-distributed appearance. To support the internalization of AT-RITC/CPP and its membrane binding, HEK293T/17 cell membranes were additionally counterstained with fluorescein-labelled choleratoxin B (CTB) (panel g). The overlay image is shown in panel h. As can be shown, both dot-like and homogeneous complex distributions are located mostly inside the cell suggesting a successful uptake and AT/CPP complex seems to be only to some extent attached to the outer membrane.

The application of hCT(18-32)-k7 as drug carrier into cells strongly demands biocompatibility, meaning a high viability rate of the cells during the delivery approach. For this reason the apoptosis rate and the vitality of HEK293T/17 cells were investigated after the AT/CPP complex co-incubation for one hour as well as after 6 h (Fig. 3). The time intervals were chosen since after 1 h, the CPP-complex uptake is completed, while after 6 h CPP complexes are finally processed within cells. No influence on cell-viability could be observed. After 1 h complex-treated cells show the same high viability as untreated control cells, whereas staurosporine-treated cells show an impaired vitality rate. After 6 h complex-treated cells show even higher vitality rates than untreated cells, whereas the vitality rate of staurosporine-treated cells further impaired.

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Figure 3. The biocompatibility of the applied complexes regarding the apoptosis- and vitality rates of the HEK293T/17 cells. Apoptosis- and vitality rates of untreated, AT/CPP complex-treated and staurosporine (5 μg/ml)-treated HEK293T/17 cells are shown. The binding of annexin V-FITC as well as the PI intercalation were flow cytometrically determined after 1 h and 6 h co-incubation time. FITC-fluorescence measurable in FL2 was compensated by substraction of 21.7%. Mean and standard deviations of three independent experiments are shown.

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Inspired by these promising results, we investigated the incubation of AT-loaded peptides with immune cells. Therefore, AT-RITC complexed with hCT(18-32)-k7 was incubated with PMN that were isolated from human whole blood, and subsequently analyzed by means of flow cytometry as well as CLSM (Fig. 4). PMN were gated according to R1 (panel a). Flow cytometry studies revealed increased fluorescence intensity when cells were incubated with the complex mixture (panel b, black curve). Applying AT-RITC without hCT(18-32)-k7 as a control also results in a fluorescence intensity increase (dark gray curve, gmean: 10.4) suggesting that both, AT and AT/CPP, are capable to interact with PMN in the same amount.

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Figure 4. The interaction of the AT-RITC/hCT(18-32)-k7 complex with PMN isolated from human whole blood measured by means of flow cytometry (a, b) and CLSM (c–g). PMN are gated according R1 (a) and used for further investigations. PMN (5 × 104) were co-incubated with the stable protein-peptide complex (hCT(18-32)-k7, 4.17 mmol/l and AT-RITC, 8.3 μmol/l). The RITC-fluorescence intensity of PMN is demonstrated in b (black). As control samples the RITC-fluorescence intensity of untreated (light gray) and PMN incubated with AT-RITC (8.3 μmol/l, dark gray) is also demonstrated (b). A representative histogram of three independent experiments is shown. The binding of the complex was additionally proved by means of CLSM. PMN (5 × 104) were co-incubated with the stable protein–peptide complex (hCT(18-32)-k7, 4.17 mmol/l and AT-RITC, 8.3 μmol/l). Cells treated with AT-RITC alone operating as control and their transmission are shown in panel (c) and (d). The RITC-fluorescence intensity of treated PMN, suggesting adsorption and uptake of the AT/CPP complex, is demonstrated in panel (e). The transmission is shown in panel (f). The overlay is demonstrated in panel (g). Representative results of three independent experiments are shown. [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com.]

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To distinguish the nature of interaction between binding and uptake, CLSM investigation was performed (panels c–g) after co-incubation of PMN with a complex of hCT(18-32)-k7 and RITC-labelled AT. PMN incubated with AT-RITC alone show a significantly emphasized fluorescence intensity along the PMN membrane (panel c, transmission image is shown in panel d). Incubating PMN with the AT-RITC/CPP complex instead resulted an in randomly dot-shaped as well as homogeneous staining of the whole cell body (panel e, transmission image is shown in panel f). According to those results, it can be assumed that the AT complex can enter the cells while AT alone is mainly capable in binding to the cellular membrane. The next attempt was to test the elastase inhibitory activity of AT after co-incubation of the AT/CPP complex with PMN (Fig. 5). Therefore, HLE activity of untreated, but (with phorbolester) stimulated PMN was measured and set to 100 %. Phorbolester stimulates the release of HLE (amongst other proteases) from intracellular granules and allows the investigation of the inhibitory influence of the AT carriers. PMN incubated with hCT(18-32)-k7 alone and subsequent phorbolester stimulation showed only a slightly enhanced HLE activity (108% ±2%) compared to the phorbolester-treated control PMN. The incubation of PMN with AT alone and the subsequent phorbolester stimulation caused an HLE inhibition of about 15% to 85% ± 3 %. In contrast, the application of complexed AT caused a further decreased HLE activity of stimulated PMN to 48% ± 10% revealing the increased uptake and successful AT processing of the complexes compared to controls.

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Figure 5. AT functionality after complex—PMN co-incubation. The AT functionality was tested by means of its capability to inhibit HLE which is released after PMN stimulation. PMN (3 × 105), untreated, treated with hCT(18-32)-k7, AT or the complex were stimulated with PMA (0.1 μmol/l). In addition, PMN supernatants collected from PMN (3 × 105) stimulated with PMA (0.1 μmol/l) were incubated with hCT(18-32)-k7, AT or the complex. HLE substrate MeOSucc-Ala-Ala-Pro-Val-p-nitroanilide (1 mmol/l) was added to the cell supernatant and HLE activity was measured at 410 nm. Means and standard deviations of three independent experiments are shown.

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Furthermore, exclusively the supernatants of phorbolester stimulated PMN were incubated with hCT(18-32)-k7, AT, and the complex mixture. The idea here was to compare the intracellular and extracellular inhibiting effect of complex-transported AT. The supernatant results show a slight increase after HCT treatment which is similar to the previously shown intracellular effect (125% ± 9%). However, the HLE activity in supernatant is significantly decreased (10% ± 1%) after AT/complex application. The difference in efficiency compared to intracellular application may be caused by the overall accessibility of complexed AT to HLE in supernatant, whereas complexed AT within the cell is only partially located within (phago)lysosomes and, therefore, exposed to granular HLE (see also Fig. 4e).

As parameter for the biocompatibility of the favoured drug delivery system, the apoptosis rate and the vitality of PMN were investigated after the AT/CPP complex co-incubation for 1 and 6 h according to the experiments with HEK293T/17 (Fig. 6). As already proven with HEK293T/17 no influence on cell-viability could be observed. After 1 h complex-treated cells show a marginal increase in cytotoxicity compared to untreated PMN, whereas staurosporine-treated cells show a higher amount of annexin V-FITC positive cells. After 6 h complex-treated cells show even less influence than untreated PMN revealing a similar behavior than after HEK 293T/17 treatment.

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Figure 6. The biocompatibility of the applied complexes regarding the apoptosis- and vitality rates of the PMN. Apoptosis- and vitality rates of untreated (a, d), AT/CPP complex-treated (b, e), and staurosporine (5 μg/ml)-treated PMN (c, f) are shown. The binding of annexin V-FITC as well as the PI intercalation were flow cytometrically determined after 1 h (a–c) and 6 h (d–f) co-incubation time. FITC-fluorescence measurable in FL2 was compensated by substraction of 21.7%. Representative results of three independent experiments are shown.

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To directly correlate the AT/complex appearance in the PMN (dot-like or homogenous distributed) with cell compartments, FITC-lysotracker staining of endo-/phagolysosomes in correlation to the localization of RITC-labelled carriers was performed. The staining of acidified compartments (Fig. 7a, green fluorescence) resulted in a partial co-localization of the RITC-labelled AT/hCT(18-32)-k7 complexes in PMN (red fluorescence, panel c), which indicates a fusion of lysosomes/primary granules and endo-/phagosomes and underlines the role of endolysosomal uptake in hCT-mediated drug delivery (Fig. 7d). In addition, a high amount of RITC-AT could be found in the cytoplasm suggesting an efficient escape of the protein out of the vesicles after endosomal uptake or a direct entry into the cell supporting the findings of a lower, but sufficient inhibitory efficiency towards intracellular then extracellular HLE. The transmission image is shown in panel b.

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Figure 7. The partial colocalization of the AT-RITC/hCT(18-32)-k7 complexes and lysosomes in PMN suggests an at least partial endo-/phagocytotic uptake. PMN (5 × 104) were co-incubated with the stable protein–peptide complex (hCT(18-32)-k7, 4.17 mmol/l and AT-RITC, 8.3 μmol/l) for 3 h at 37°C. Lysosomal staining was performed by means of Lysotracker Green (50 nmol/l) for 1 h at 37°C (a). Fluorescence measurements were performed by means of CLSM. RITC-stained AT/hCT(18-32)-k7 complexes are visible in (c). Transmission and overlay are shown in (b) and (d). A representative result of three independent experiments is shown. [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com.]

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In general, these results indicate an enhanced AT efficiency in PMN when used as a complex rather than as single molecule, whereas the efficacy in supernatant is comparable for both forms due to a good accessibility of free as well as complexed AT towards HLE.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. LITERATURE CITED

Chronic inflammation constitutes a challenging accessory symptom of several diseases of civilization. Although chronic processes are responsible for numerous disturbing effects, current medication is rather unsatisfying due to the need of high doses as well as the induction of severe side-effects (10). Therefore, the aim of this study was the direct CPP-mediated AT administration to inflammatory cells, e.g. PMN. We intend the attenuation of the damaging effects of proteases, as HLE. HLE is supposed to be inhibited by AT/CPP complexes when [1] it is already released as well as [2] at its place of origin, inside specific cell compartments (granules). Subsequently, the CPP-mediated AT transfer leads to an increased intra- and extracellularly AT concentration in order to reconstitute the disturbed protease/antiprotease balance and to attenuate the protease-mediated tissue degradation. The advantage of a locally applied drug carrier based medication avoids negative effects which may occur during systemic drug usage. The local restriction further allows a low-dose drug application preventing patients from unnecessary burden.

The results presented in this study highlight the only marginal binding of free AT to HEK293T/17 cells as well as high binding to PMN membranes, but without significant uptake, whereas a pronounced uptake of complexed AT into HEK293T/17 and PMN occurs. The clear difference between the cell's surface binding behavior of AT/complex may be mostly motivated by already released and surface bound HLE due to the strong AT-HLE interaction rather than charge-associated complex binding to the membrane. Second, the AT/CPP distribution in the cell correlates partially with the lysosomal staining indicating an at least partial endolysosomal uptake and processing. The localization of AT-RITC in other cell compartments may result from early release from endo-/phagolysosomes into the cytoplasm or from a direct cell penetration of the complex. Two fluorescence staining patterns were observed by CLSM: on the one hand, there is a clear dot-like distribution of the complexed AT co-localized with acidic compartments strongly suggesting an endo-/phagocytotic uptake mechanism, and on the other hand, labeled AT can also be found randomly/evenly distributed in cells. Beside early endosomal escape, this staining pattern also indicates an endothelial uptake by penetration. Both uptake mechanisms are conceivable to coexist, whereas an endo-/phagocytotic pathway for the intended delivery approach are preferable to get access to HLE-containing primary granules. At third, another important finding is the enhanced HLE inhibitory activity of CPP-complexed AT in the cell,additionally suggesting an at least partial phagocytotic uptake of the serpin by PMN. This result is promising for an intended advanced therapy. And fourth, within the optimal incubation time frame (1–6 h) regarding CPP uptake, no cytotoxic effect of AT/CPP could be found.

From our findings, we can deduce a mechanism of AT/CPP processing which is schematically demonstrated in Figure 8. Here, tissue disturbing HLE activity can be blocked in two different ways: On the one hand, HLE can be inhibited outside of PMN (1) and on the other hand, HLE activity can already be inhibited inside specific cell compartments before its release into the extracellular space (2, 3). First, in interaction with cells AT/CPP complexes adhere to PMN membranes by means of positively charged peptide residues or via AT interaction with surface-bound HLE (1). AT-mediated HLE inhibition can be performed either at the surface of PMN membranes or freely in extracellular space. The second HLE inhibitory pathway will be performed intracellularly. CPP-mediated phagocytotic AT uptake (3) by immuno-reactive cells facilitates the contact of HLE stored in azurophilic granules and AT during the fusion of endosomes and azurophilic granules which occurs already during the first hour of co-incubation as could be shown by pH measurements (18).

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Figure 8. The schematic sketch of the suggested complex formation between AT and the CPP hCT(18-32)-k7 based on electrostatic interaction and the cellular uptake mechanism which is supposed to be both, endothelial as well as an endo-/phagocytotic pathway. Proteases are released by PMN during stimulation and subsequently bound to membrane surface areas. AT/hCT(18-32)-k7 complexes are capable to inhibit HLE (1) at the PMN surface by formation of enzyme/inhibitor complexes and (2) inside endo/phagolysosomes after endo-/phagocytotic uptake and fusion which gains access to internal HLE. Here, engulfed AT/hCT(18-32)-k/ complexes will gain contact with HLE once phago- and lysosomes are fused to become a phagolysosome. Consequently, enzyme/inhibitor complexes can be already formed within the cellular body. [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com.]

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Membrane adherence of the complex structure is encouraged by the highly cationic residues surrounding AT in the complex independent of the cell type. Due to the used CPP excess (500-fold), significant membrane adsorption should be supported by means of high amounts of cationic residues. Thus, to avoid negative effects due to the highly cationic character of the AT/CPP complex, a local application is favored. The interaction with cell membranes could give the signal for the endo-/phagocytotic, e.g. lipid raft dependent, uptake. Until now, the specific mechanism is not clear. Purified AT by itself was described to be internalized by lung endothelial cells in a clathrin-dependent way to perform an anti-apoptotic role inside the lung endothelium (19). In addition, macrophages (human monocyte-derived macrophages and primary rat alveolar macrophages) also exhibit AT internalization. Target enzyme/AT complexes are supposed to be internalized via endocytosis. A serpin/enzyme complex receptor has been reported to be responsible for endocytosis of AT/HLE complexes (19, 20). Presumably a receptor-mediated serpin/enzyme complex internalization plays a role during AT endocytosis and HLE inhibition at PMN surfaces.

Regarding biocompatibility hCT(18-32)-k7 can be considered as a promising tool for noninvasive cellular import of the cargo. In general, CPPs show a lack of cytotoxicity (11). Although an over-excess of the highly cationic peptide hCT(18-32)-k7 (500 fold) was applied, no cytotoxic or apoptosis-inducing effects could be observed in both cell types. This highly biocompatible characteristic makes it suitable for in vivo application. This is in agreement to recent findings concerning the cytotoxic profile of hCT(18-32)-k7 when incubated with different cell lines (21).

A local application of the complex will further decrease potential negative side-effects avoiding the contact with most of other cells and tissues as well as restrict the drug influence to a very small body area.

Among numerous advantageous features as good penetration into tissues, the low CPP resistance against proteolytic enzymes—in some cases a drawback, but here preferable—allows a rapid clearance from the body by enzyme degradation. Especially concerning the basic sequence (k7), sensitivity to trypsin-like enzymes is described resulting in degradation within 24 h. However, due to steric hindrance the transported cargo may also play a role regarding the carrier peptide stability (13).

A further challenging point of the complex application in PMN for the inhibition of HLE is the slightly enhanced HLE activity after hCT(18-32)-k7/PMN co-incubation and subsequent stimulation of the cells with phorbolester. This slightly enhanced cell activation may be based on the highly cationic character of the peptide. But, as can be seen from the complex/PMN co-incubation, this CPP-mediated HLE activity increasing effect is completely abolished by AT-inhibiting effects and therefore, can be neglected for the complex application.

Although, further studies have to be performed in future in order to improve the application of AT as well as other active agents in immunologic cells, the achieved uptake of the hCT(18-32)-k7 complexed AT by PMN as well as the maintained functionality seem to be the first steps for an aimed modern and gentle therapy method in chronic inflammation. The intended therapy form could also be a further development of the currently only available specific therapy for AT deficiency. The only effective current treatment for patients with chronic obstructive pulmonary disease (COPD)/AT deficiency and cystic fibrosis is replacement therapy with purified AT from human plasma (22–24). Other therapeutic options are augmentation by gene therapy and gene repair, but they are still under development. The here described approach could decrease the so far cost-effectiveness due to the higher HLE inhibitory activity of AT after the binding and uptake by PMN.

CONCLUSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. LITERATURE CITED

As we could show in this study, complex formation of AT by means of a potential drug carrier, hCT(18-32)-k7, its uptake by several cells and functionality maintenance are possible, a direct CPP-mediated AT administration into immuno-reactive cells in order to attenuate protease tissue destruction in chronic inflammatory loci could become a new strategy in therapy. The convenience of this method, making it more interesting than others, is the high biocompatibility of CPPs, its low cytotoxicity, and its applicability for a variety of other active agents establishing the potentiality to combine the application of several drugs or effectors to enhance the positive effects. hCT(18-32)-k7 complexed with anti-inflammatory agents could constitute a non-invasive, gentle treatment method in chronic processes.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. LITERATURE CITED
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