Mechanisms of Internalization of Maltose-Modified Poly

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Mechanisms of internalization of maltose-modified poly(propylene imine) glycodendrimers into leukemic cell lines Maciej Studzian, Aleksandra Szulc, Anna Janaszewska, Dietmar Appelhans, #ukasz Pu#aski, and Barbara Klajnert-Maculewicz Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00046 • Publication Date (Web): 17 Apr 2017 Downloaded from http://pubs.acs.org on April 18, 2017

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Mechanisms of internalization of maltose-modified poly(propylene imine) glycodendrimers into leukemic cell lines Maciej Studziana,b*, Aleksandra Szulca, Anna Janaszewskaa, Dietmar Appelhansc, Łukasz Pułaskib,d, Barbara Klajnert-Maculewicza,c* a

Department of General Biophysics, Faculty of Biology and Environmental Protection,

University of Lodz, 141/143 Pomorska St., 90-236 Lodz, Poland; b

Department of Molecular Biophysics, Faculty of Biology and Environmental Protection,

University of Lodz, 141/143 Pomorska St., 90-236 Lodz, Poland; c

Leibniz Institute of Polymer Research Dresden, Hohe Str. 6, 01069 Dresden, Germany;

d

Laboratory of Transcriptional Regulation, Institute of Medical Biology PAS,

Lodowa 106, 93-232 Lodz, Poland

ACS Paragon Plus Environment

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Abstract Poly(propylene imine) dendrimers of 4th generation partially modified with maltose (open shell structure, PPI-m OS) have been proposed as carriers for nucleotide anticancer drugs. The aim of this work was to provide basic insight into interactions between fluorescently labeled PPI-m dendrimer and two distinct leukemia cell models: CCRF-1301 lymphoid cell line and HL-60 myeloid cell line. We applied qualitative confocal imaging and quantitative flow cytometry, as well as trypan blue quenching and pharmacological inhibition, to investigate the course, kinetics and molecular mechanisms of internalization of nanoparticles. CCRF-1301 cells take up glycodendrimer macromolecules via a relatively slow, adsorptive endocytosis process which is cholesterol-dependent, clathrin- and caveolin-independent and is not followed by recycling or exocytosis. Morphological features of this phenomenon point to the involvement of aggregationinduced cell polarity changes (capping). In HL-60 cells, internalization is very fast, independent of binding to the cell surface and proceeds from the fluid phase via a classical clathrin-dependent mechanism, ending up in an endolysosomal compartment from which it is not further released. This substantial difference in internalization rate and mechanism between two cell types has important repercussions for potential application of this class of glycodendrimers as drug delivery agents.

Keywords: endocytosis; dendrimers; fluorescence; leukemia


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Introduction Dendrimers are highly branched polymeric macromolecules of nanoscale size. Due to their unique structure they constitute an interesting material with a number of potential biomedical applications 1, 2. Regular and repetitive monomers called dendrons diverge radially from the core, i.e. central polyvalent moiety. As a consequence, dendrimers possess the ability to bind drug molecules inside their structure as well as numerous functional peripheral groups decorating their surface and providing desired properties such as: increased solubility or higher bioavailability and specificity towards particular therapeutic targets

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. Moreover, pharmacologically active

molecules, while not being permanently conjugated to the vehicle, may still be successfully encapsulated in the interior of the dendritic moiety or may dynamically attach to the surface functional groups 6. Such drug-dendrimer formulations are usually characterized by much higher stability than other potential drug carrier systems like liposomes or micelles 7, 8. Over the last couple of years many different dendritic scaffolds such as: viologen-phosphorus dendrimers, lactobionic acid-modified PAMAM dendrimers or oligosaccharide-modified hyperbranched poly(ethylene imines) were considered as potential carriers for chemotherapeutic agents for cancer treatment

9-11

. One of the most promising scaffolds is poly(propylene imine)

(PPI). PPI dendrimers are composed of the central 1,4-diaminobutane molecule and branched poly(propylene imine) chains

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. Abundant external and internal amino groups are responsible

for their overall strong positive charge at physiological pH which allows them to form longlasting electrostatic interactions with many anionic drug molecules such as nucleotide analogs or antisense oligodeoxynucleotides 13, 14. On the other hand, since unmodified PPI nanoparticles are highly cytotoxic and hemolytic

15, 16

, it is a prerequisite to weaken the positive charge on their

surface in order to obtain more biocompatible nanocarriers 17, 18. Multiple PPI dendrimer surface


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modifications have been studied. Among others, modifications with sugar moieties have been found to not only significantly lower the innate cytotoxic properties of this class of dendrimers 19-21

, but also in many cases improve specificity of their interactions with selected cell types 22.

For example, macrophages were specifically targeted by modifications with sialic acid and mannose residues 23. In our previous studies we have proven that PPI dendrimer of 4th generation partially modified with maltose (open shell structure, PPI-m OS), thanks to the moderate positive charge and internal cavities of optimal size, is able to efficiently form complexes with cytarabine triphosphate (Ara-CTP) at pharmacologically relevant concentrations of the drug

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. Such

complex of PPI-m OS glycodendrimer with the anionic nucleotide may be an efficient way to deliver active form of the drug directly to tumor cells, potentially bypassing a number of factors frequently limiting effectiveness of the chemotherapy such as: multidrug resistance, low biodistribution or fast metabolism of the drug 25. Since the 4th generation glycodendrimer forms optimal drug complexes and has significantly lower cytotoxicity than its unmodified counterpart 20

, we chose it for the present study.

The key step to further advance towards successful applicability of this maltose-modified PPI dendrimer as a drug delivery vector is a thorough analysis of its uptake by specific types of cancer cells. Although there is a significant body of literature concerning internalization and trafficking routes of diverse dendritic scaffolds and glycosylation-mediated targeted carriers in various cell lines

26, 27

, still little general conclusions or predictions can be made. Numerous

studies have shown that the pathway of intracellular uptake is not only dependent on the shape, size or surface charge of the particular nanoparticle 28, but it also largely differs between various cancer cell lines. Cellular uptake of unmodified PPI dendrimers which are relatively cytotoxic


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has been investigated only in conjunction with their use as siRNA delivery agents in ovarian cancer cells and was found to be non-specific

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. Internalization mechanisms of maltose-

modified PPI dendrimers have so far only been studied in melanoma cells and turned out to be manifold 30, again supporting the argument that the relevance of particular dendrimer for use in cancer chemotherapy must be substantiated for a specific type of cancer cells. The aim of this work was to provide basic insight into interactions between fluorescently labeled maltose-modified PPI dendrimer of the 4th generation and two distinct leukemia cell models. It is known that nucleoside and nucleotide analogue drugs that might potentially be transported into malignant cells by partially maltose-decorated PPI dendrimers are default therapeutics in treatment of acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL) and lymphomas

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. Therefore, representative T-lymphocytic (CCRF-1301) and myeloid (HL-60)

leukemia cell lines were selected as test subjects for this study.


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Materials and method Preparation of fluorescein-labeled glycodendrimer Open shell poly(propylene imine) dendrimer of the 4th generation conjugated with fluorescein molecule (PPI-m-FITC OS G4) and with primary amino surface groups partially decorated with maltose (degree of modification ~30%) was synthesized and characterized as previously described

32, 33

. Briefly, 4th generation poly(propylene imine) dendrimer was purchased from

SyMoChem (Eindhoven, Netherlands). This dendrimer is spuriously commercialized as “5th generation” and is sometimes described (e.g. 30) as such – a recent review explains the source of this confusion

17

. This compound was functionalized with maltose residues by reductive

amination in the presence of maltose in sodium borate buffer using a borane•pyridine complex as reducing agent. Subsequently, fluorescein isothiocyanate in DMSO was added dropwise to a water solution of PPI-m OS G4 at final stoichiometry 2:1. After completion, unbound dye was removed by size exclusion chromatography and dialysis against water (until fluorescence was undetectable in the dialysate). Molecular weight of the final product (MW 20773 g/mol) was ascertained by established mass spectrometry and 1H NMR approach

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. Stock solution of the

dendrimer was prepared by lyophilization and resolubilization in PBS, it was filter-sterilized and kept at 4°C until use.

Other materials Dynasore (dynamin-specific inhibitor) was bought from Calbiochem (USA). Pitstop 2, inhibitor of clathrin-mediated endocytosis, was purchased from Abcam (UK). DiL, lipophilic plasma membrane dye, was acquired from Biotium (USA). All other chemical inhibitors and reagents were purchased from Sigma-Aldrich (USA).


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Cell culture Human acute myeloid leukemia cell line HL-60 was purchased from ATCC (USA). Human acute lymphoblastic T cell leukemia cell line CCRF-1301 was obtained from Banca Biologica e Cell Factory (Italy). Both cell lines were cultured at 37°C (humidity 95%, CO2 5%) in Roswell Park Memorial Institute medium (RPMI-1640, Sigma Aldrich) containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma Aldrich). Cell counting and viability assessment was routinely performed on automated cell counter (Countess, Life Technologies; USA). Cytotoxicity assay To verify the cytotoxicity of PPI-m-FITC OS G4 glycodendrimers, resazurin assay

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was

performed. Cells were seeded on black 96-well plates at a density of 1.5 × 104 (CCRF-1301) or 2 × 104 (HL-60) cells per well and treated with increasing concentrations of dendrimer ranging from 0.05 µM to 2 µM for 24 and 48 hours. Following the incubation, resazurin solution was added to the cells to final concentration of 12.5 µg/ml and plates were incubated for 2 hours at 37°C in darkness to allow conversion of resazurin to resorufin. Fluorescence of metabolized resazurin was measured with a fluorescence microplate reader (Fluoroskan Ascent, λexc = 530 nm, λem = 590 nm). Cell viability was presented as percent of value for the untreated control. The measurements were repeated in four independent experiments and data for equally treated cells were derived from six wells for each microplate. Flow cytometry measurements In all treatments, 5 × 105 cells of both leukemia cell lines were seeded per each experimental point in 1 ml of complete growth medium in 24-well plate. In dendrimer uptake study, PPI-mFITC OS G4 glycodendrimer was added to cell suspension to get a final concentration of 0.1 µM


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or 1 µM and cells were incubated in normal growth conditions for 1, 3, 6, 24 and 48 hours. Immediately after, cells were placed on ice in darkness until FACS analysis. Intracellular fate of PPI-m-FITC OS G4 was studied analogously. Cells were loaded with 0.1 µM (CCRF-1301) or 1 µM (HL-60) PPI-m-FITC fluorescent glycodendrimer for 24 hours. Thereafter, cells were briefly centrifuged (200 × g, 5 min), resuspended in fresh medium and their total/intracellular fluorescence was measured at the initial time point (0h). Subsequently, cells were cultured at 37°C for 1, 3, 6, 24 and 48hours and after that, they were placed on ice in darkness until FACS analysis. Flow cytometry measurements were performed on LSR II system (Becton Dickinson, USA). In each experiment 10000 events were counted after gating of viable cells and median of fluorescence intensity in FITC channel was calculated for each population. As a control, untreated cells were measured similarly and median of auto-fluorescence intensity was calculated and subtracted from each experimental result in parallel for each cell line. In order to measure intensity of fluorescence of internally localized dendrimer, cells were pre-incubated for 5 min on ice with equal volume of 0.4% trypan blue solution and analyzed immediately after. All data were analyzed with FACSDiva software (Becton Dickinson, USA). Confocal microscopy imaging Cells were treated for indicated times with PPI-m-FITC OS G4 dendrimer at 0.1 µM or 1 µM concentration. Following treatment, cells were seeded at a density of 1 × 104 cells/well (in 100 µl of full growth medium) on SensoPlate (Greiner Bio-One; USA) 96-well thin-glass bottomed plate coated previously with poly-L-lysine. After 10 minutes of sedimentation at 37°C (or at 4°C when endocytosis was inhibited by low temperature) cells were washed once with PBS and imaged immediately (Figure 5 and Figure 6) or after additional staining (Figure 2). When imaged


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immediately, pictures were taken in the same field of view before and after addition of equal volume (100 µl) of 0.4% trypan blue solution. Trypan blue, a cell membrane-impermeable dye, was added for dual effect: to quench fluorescence of extracellular and membrane-bound dendrimer and to act as a fluorescent dye binding to cellular proteins (exclusively plasma membrane proteins in viable cells and the entire cell in permeabilized cells). For staining of cell nuclei and plasma membrane, living cells were first incubated at 37°C with 5 µM Hoechst 33342 dye for 20 min. Subsequently, cells were cooled to 4°C to inhibit endocytosis, stained for 2 min with plasma membrane dye DiL (diluted 1:200), washed once with PBS and imaged immediately afterwards. Cells were imaged using LSM780 microscope equipped with Plan-Apochromat 63x/1.4 Oil DIC M27 objective and In Tune™ tunable excitation laser system (Carl Zeiss; Germany). Fluorescence of PPI-m-FITC OS G4 was visualized with 490 nm excitation and in 500 nm – 545 nm emission range. Fluorescence of trypan blue-protein conjugate was excited with In Tune™ laser at 553 nm and detected in 580 nm – 740 nm emission range. Inhibition of endocytosis Cells were cooled to 4°C or pre-treated for 30 min with endocytosis inhibitors: 80 µM dynasore, 10 µM chlorpromazine, 20 µM triflupromazine, 30 µM Pitstop 2, 7.5 µM filipin complex, 25µM nystatin, 200 µM genistein, 10 µM PP2 or 100 nM wortmannin. Cells were then further incubated for 1 h or 24 h with 0.1 µM PPI-m-FITC OS G4 dendrimer in the presence of respective inhibitor and analyzed by confocal microscopy or flow cytometry as described above. Statistical analysis All data are presented as mean ± S.E.M. and asterisks are used to mark statistical significance of differences, with n values given in figure legends. The differences between two groups were


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analyzed by Student’s two-tailed t test. For multiple group comparison one-way ANOVA test was applied and Tukey’s post-hoc test was used for pairwise difference. The value of p < 0.05 was considered statistically significant.


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Results Poly(propylene imine) glycodendrimer used for this study (PPI-m OS G4) is moderately cationic in physiological conditions (only ~30% of the surface primary amino groups are conjugated to the sugar moiety), but was previously described to be harmless to a number of human cell lines 30. To ensure that fluorescein-modified conjugate (PPI-m-FITC OS G4) studied here is not inherently toxic for human cellular leukemia models, we have first examined its effect on cell viability of acute myeloid (HL-60) and T-lymphoblastic (CCRF-1301) leukemia cell lines. As expected, neither 24 h nor 48 h exposure had any detrimental impact on cell survival of either cell line (Fig. 1). Measured viability was always higher than 90% and observed fluctuations were not statistically significant for dendrimer concentration up to 2 µM.

Figure 1. Cytotoxicity of PPI-m-FITC OS G4 dendrimer in leukemia cell lines. CCRF-1301 (A) and HL-60 (B) cells were treated with dendrimer for 24 h (regular line, closed circle) or 48 h (dashed line, empty triangle). Cell viability was then assessed with resazurin assay and is presented as percent of untreated control; data shown as mean ± S.E.M., n=4.


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Next, we have set out to investigate whether PPI-m-FITC OS G4 dendrimer is able to interact with model leukemia cells and to translocate to their interior when used at non-toxic concentrations. For this purpose, HL-60 and CCRF-1301 cells were incubated with two welltolerated concentrations of the macromolecule (0.1 µM and 1 µM) for a 2-hour period that is generally sufficient for cells to internalize a cargo molecule

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. Confocal microscopy images of

the cells were taken after subsequent counterstaining of cell nuclei and plasma membrane and are shown in Figure 2. Both cell lines bind fluorescently modified nanoparticles, although clear differences in signal intensity and location are observed.


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Figure 2. Confocal imaging of human leukemia cells treated with PPI-m-FITC OS G4 dendrimer. CCRF-1301 and HL-60 cells were incubated for 2 h with 0.1 µM or 1 µM PPI-m-FITC OS G4 dendrimer (green pseudocolor). Subsequently, cell nuclei were visualized with Hoechst33342 dye (depicted as blue pseudocolor), plasma membrane was stained with DiL dye (red pseudocolor) and cells were imaged immediately afterwards. Characteristic clumping of PPI-mFITC OS G4 dendrimer was observed on the surface of CCRF-1301 cells, the “caps” formed by the nanoparticle are indicated by arrows.

Interestingly, in CCRF-1301 cells after 2 h treatment, maltose-decorated dendrimer was predominantly colocalizing with the cell membrane staining and almost no intracellular signal could be detected. Additionally, characteristic cell polarization was observed in this line, with dendrimer being concentrated on one edge of the cell, forming caps (Fig. 2 indicated by arrows, upper panel). HL-60 cells, on the other hand, accrue significantly more of the nanoparticle which is contained in defined, uniformly distributed endosome-like structures inside the cell. In order to quantitatively examine cellular adhesion as well as internalization, we have decided to incubate cells with the same two concentrations of fluorescently modified dendrimer and to measure cellular fluorescence at different time points. Moreover, to discriminate between total and internalized fluorescence signal, for each time point the same population of cells was analyzed with flow cytometry without and with addition of trypan blue, used for quenching external and membrane-bound fluorescence in intact cells (Fig. 3) 36, 37.


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Figure 3. Cell adhesion and internalization of PPI-m-FITC OS G4 dendrimer. CCRF-1301 cells (A, B) and HL-60 cells (C, D) were incubated with low concentration (0.1 µM; A, C) or high concentration (1 µM; B, D) of PPI-m-FITC OS G4 dendrimer. For each time point both total (without trypan blue) and intracellular (after trypan blue quenching) median fluorescence intensity (MFI) in FITC channel was analyzed by flow cytometry; data presented as mean ± S.E.M., n=3; asterisks denote statistically significant difference from the preceding time point (p

Mechanisms of Internalization of Maltose-Modified Poly

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