Mechanistic Insight into Cell Growth, Internalization, and Cytotoxicity of

Dec 28, 2009 - ... would like to acknowledge funding from Starpharma Holdings Pty. ... Ms. Renee Schulz, Dr. Paul Nielsen, and Dr. Raman Sharma are ...
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Biomacromolecules 2010, 11, 382–389

Mechanistic Insight into Cell Growth, Internalization, and Cytotoxicity of PAMAM Dendrimers Srinivas Parimi,† Timothy J. Barnes,† David F. Callen,‡ and Clive A. Prestidge*,† Ian Wark Research Institute, ARC Special Research Centre for Particle and Material Interfaces, University of South Australia, Mawson Lakes, SA-5095, Australia, and Breast Cancer Genetics Group, Discipline of Medicine, University of Adelaide and Hanson Institute, Institute of Medical and Veterinary Science, SA-5000, Australia Received September 4, 2009; Revised Manuscript Received December 10, 2009

We report on the role of PAMAM dendrimer concentration and generation (G2, G4, G6) on cell growth and cytotoxicity in HEK293T and HeLa cell lines and make comparisons with dendrimer-induced leakage from liposomes to probe the mechanisms in action. Specifically, we observed a striking transition from cell growth enhancement to a reduction in cell viability at a critical PAMAM dendrimer concentration, that is, ∼500 nM. Confocal microscopy studies show evidence of a transition from cell membrane adhesion to cell internalization and cell nucleus interaction at equivalent dendrimer concentrations. A dendrimer concentration window of 500-700 nM was identified for effective cell internalization without significant cytotoxicity. Though liposome leakage correlated with cytotoxicity, no quantitative agreement was observed, that is, cells are 100 times (based on surface coverage) more resistant to dendrimers than liposomes. These findings have significant implications in the design of effective drug/gene delivery vehicles based on dendrimers.

Introduction Polyamidoamine (PAMAM) dendrimers are commercially available, widely used synthetic hyperbranched polymers with a high degree of monodispersity, polyvalency, and controlled molecular architecture.1,2 These characteristic features have generated considerable interest in their use as drug2–5 and gene delivery vehicles.6–8 However, progress toward such application has been restricted due to limited knowledge of the influence of dendrimer concentration and generation on the mechanisms of cell membrane transport and cellular uptake. Biological cell membrane transport is considered a crucial aspect of drug or gene delivery.3 The presence of various physicochemical and membrane associated intracellular barriers hamper intracellular delivery or transport of therapeutics across biological barriers.4 Dendritic polymers have been widely investigated as vehicles to overcome these barriers via either paracellular and transcellular pathways.5 Recent studies by Saovapakhiran et al.6 and Kitchens et al.7,8 reported that a clathrin mediated endocytosis mechanism contributed to the transport or internalization of PAMAM dendrimers in human colon adenocarcinoma (HT-29) and in Caco-2 cell lines, respectively. Dendrimer surface composition has a dramatic effect on cell internalization and trafficking properties.6,9 Physical adsorption10 followed by formation of nanoscale holes in the bilayer11,12 are considered critical in controlling cell membrane permeation of dendrimers. The physicochemical properties of dendrimers such as size, molecular weight, surface groups,13 dendrimer type,9 surface chemistry,14 generation,15 and concentration16 are reported to dictate their ability to translocate across the lipid bilayer. High levels of dendrimer internalization are also associated with cytotoxicity, as demonstrated for arginine- and ornithine-conjugated PAMAM dendrimers and * To whom correspondence should be addressed. Fax: 61-8-83023683. E-mail: [email protected]. † University of South Australia. ‡ University of Adelaide and Hanson Institute.

Caco-2 cells.15,16 In contrast, a recent study by Tziveleka et al.17 demonstrated limited cytotoxicity and significant transfection for a G4 poly (propylene imine) dendrimer into HEK293 and COS-7 cell lines due to the accumulation of guanidinium groups at the dendrimeric surface and enhanced penetrating ability of guanidinylated dendrimers. It is clear that more systematic studies are required to establish the relationship between dendrimer concentration/generation and cell internalization/cytotoxicity. Liposomes are widely exploited as building blocks for artificial membranes, mimicking membrane- drug interactions or drug delivery processes.18 Furthermore, their nontoxicity19 and close analogy to cells14,20 has led to their use as effective carrier systems for intravenous delivery of drugs. Studies of dendrimer interactions with liposomes can be useful in mimicking cellular interactions.21 A wide range of dendrimer-liposome interaction studies have shown the occurrence of structural changes to the lipid bilayer and increased susceptibility of nonlamellar phospholipid phases to rupture.1,6,23–26 For example, higher generation PAMAM have enhanced interactions with 1,2dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes,22,23 while hydrophobically modified (50%-C12) PAMAM dendrimers increased their permeation into phospholipid bilayers.15 Investigations of liposome membrane transport and colloidal stability induced by dendrimers24–27 are also useful in gaining insight into their bioactivity. Tsogas et al.14 reported that an optimum balance between the binding strength of guanidinium with the phosphate groups and the degree of hydrophilicity of guanidinylated dendrimers is required for the transport to the liposomal core. In this study, we investigated PAMAM dendrimer induced growth and cytotoxicity in HeLa and HEK293T cell lines, as a function of dendrimer generation and concentration. Internalization of the FITC-labeled G6 PAMAM following exposure to HeLa cells was visualized by laser scanning confocal microscopy and the results correlated with the cell growth and

10.1021/bm9010134  2010 American Chemical Society Published on Web 12/28/2009

Mechanistic Insight of PAMAM Dendrimers

cytotoxicity. Phospholipid bilayer disruption by PAMAM dendrimers is monitored by leakage of calcein from phospholipid liposomes. Results from these studies provide mechanistic insight into dendrimer interactions with cells.

Experimental Section Materials. G2, G4, and G6 PAMAM dendrimers (as aqueous solutions), fluorescent probes fluorescein (5) isothiocyanate (FITC), Calcein, (N-[2-hydroxyethyl] piperazine-N1-[2-ethanesulfonic acid]) hemisodium salt (HEPES), and 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI) mounting media were purchased from SigmaAldrich (Castle Hill, NSW, Australia) and used as received. Lecithin (VWR International Ltd., Poole, U.K.) was used as received. Calcium chloride dihydrate (B.D.H Chemicals, Kilsyth, Australia), sodium chloride (Chem-Supply, Australia), and phosphate-buffered saline (PBS, Invitrogen, Victoria, Australia) were used in buffer preparations in ultra pure water with a resistivity of 18.2 MΩ. Cell Culture and Treatment. Cells lines (HeLa, HEK293T) obtained from ATCC (American Type Tissue Collection, Rockville, MD) were cultured in a Dulbecco’s modified Eagle’s medium (DMEM, Sigma) supplemented with 10% fetal calf serum (FCS, Sigma Aldrich) in a T25 culture flasks (Nunc, Denmark) and were incubated at 37 °C in a humidified atmosphere containing 5% CO2. Cells were also regularly passaged by trypsinization with 0.1% trypsin in PBS (pH 7.2). In Vitro Cell Growth and Cytotoxicity. In vitro cytotoxicity studies toward HeLa and HEK293T cells were performed. Briefly, both type of cells were seeded at a density of 2 × 104 cells/100 µL into a 96well plate and maintained at 37 °C in an atmosphere of 5% CO2 and 95% relative humidity. Cell culture media DMEM was supplemented with 10% FCS and streptomycin (100 µg/mL). The cells were incubated for 4 h before initiating the assay. Two different types of experiments were performed. First, the trypsinized cells were washed with PBS followed by addition of DMEM and treatment with different concentrations (100, 200, 500, 700, and 1000 nM) of PAMAM dendrimers. These dendrimer coated cells were then washed by centrifugation at 2500 rpm for 5 min and the resulting cell pellets were resuspended in DMEM and aliquoted into a 96-well plate. In the second type of experiment, untreated cells were allowed to adhere to the substrate, followed by addition of different PAMAM dendrimer concentrations. After subsequent incubation for different time periods (0, 24, and 48 h), the cells were washed in PBS and replaced with fresh 100 µL DMEM media. CellTiter-Glo reagent (Promega, Alexandria, NSW, Australia) was added, and the relative cell numbers were determined. This reagent accurately measures the viable cell numbers in the culture using luciferase enzyme and measuring ATP as an indicator of metabolically active cells. The luminescent signal as measured by a Luminometer (Bio-Rad- Model 550, Australia) is proportional to the number of viable cells present in the culture. Fluorescent Labeling of PAMAM Dendrimers. FITC-labeled PAMAM dendrimers (G4 and G6) were labeled using a previously described method.8,28,29 Briefly, FITC was dissolved in acetone (5 mg/ mL) and was added to PAMAM dendrimer solutions (PBS pH 7.4) with excess FITC added. The reaction mixture was incubated in dark with stirring for 24 h. Unconjugated FITC was removed by dialysis (molecular weight cut off ) 1000 Da, Spectrum Laboratories Rancho Dominguez, CA) for 3 days against distilled water. FITC-labeled dendrimers were refrigerated ( G4 > G2) and is due to higher functional group density and more spherical conformation of G6 dendrimers. Liposomal membrane disruption and calcein leakage occurred over the dendrimer concentration range 1 and 100 nM. These concentrations correspond to surface coverage values as low as a few percent of a monolayer and are significantly lower than those required to initiate cytotoxicity in cells. It is also instructive to consider the particle size (Figure 7A) and ζ-potential (Figure 7B) of liposomes in the presence of dendrimers. PAMAM dendrimer adsorption causes charge neutralization at ∼100 nM concentration and liposome

Figure 4. Effect of dendrimer concentration and generation on (A) HeLa cell; (B) HEK293T cell line viability after 24 h incubation at 37 °C. Results are reported as mean ( standard error (n ) 3 and p < 0.05).

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Figure 5. Confocal fluorescence microscopic images of HeLa cells. (A and D) Differential interference contrast (DIC) images. (B) Treatment with 700 nM of G6-FITC; (C) 40× magnification of merged FITC/DAPI images; (E) treatment with 1000 nM of G6-FITC; (F) merged Z-section images. Note that the marked lines are cell boundaries and green fluorescence of G6-FITC, which indicate colocalization of dendrimers around the cell nucleus at higher dendrimer concentrations (scale bar: 10 µm, unless otherwise indicated).

Figure 6. Calcein leakage from liposome (SLC) after the addition of (A) different generations of PAMAM dendrimers at 100 nM (symbols: 9 G2; 2 G4; b G6). (B) Maximum calcein leakage for different generation dendrimers at various concentrations.

Figure 7. (A) Particle size; (B) zeta potential of PAMAM dendrimer-lecithin liposome complex. Each result represents the mean value of three runs.

aggregation; charge reversal and redispersion occurs at higher concentrations. At dendrimer concentrations in the range of 1 and 10 nM negligible variation in liposome particle size and ζ-potential are observed, in agreement with their lower surface coverage (Table 2B). The formation of dendrimer-

liposome aggregates or fusion of liposomes and, therefore, enhanced lipid bilayer disruption (caused by hole formation) play a major role in controlling this behavior. Findings are in good agreement with previous studies53,12 that reported the impact of PAMAM dendrimers on the disruption kinetics

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of supported DMPC lipid bilayers, that is, a dendrimer generation and concentration dependence. At dendrimer concentrations >500 nM, a plateau in the positive ζ-potential is observed, suggesting no further increase in dendrimer adsorption and corresponding suppression of dye leakage. This effect is indicative of creation of steric hindrance to vesicle aggregation, which leads to redispersion of dendrimer-liposome aggregates showing particle sizes similar to that of the initial liposomes. Findings are in agreement with Karoonuthaisiri et al.25 who reported suppression in leakage from dioleoyl-sn-glycero-3-phosphoethanolamineoleic acid (DOPE-OE) vesicles following treatment with 330 nM of G6 PAMAM dendrimers. Liposome leakage and cytotoxicity are in good qualitative agreement, but show significant quantitative differences. That is, liposome leakage occurs at concentrations more than 100 times less and equivalent surface coverages more than 4 orders of magnitude lower than was required to significantly reduce cell viability. Liposome leakage mimics cell lysis; however, the complexity of cell membranes and the selfrepairing ability of cells results in these quantitative differences. It is, however, instructive to establish what effect dendrimers have on liposomes (interfacial, colloidal, and propensity of bilayer disruption) at concentrations in the vicinity of C*, so as to gain more insight into the mechanisms of cytotoxicity and hence optimization of drug delivery and cell uptake.

Conclusion PAMAM dendrimer concentration and generation dependent cell growth and cytotoxicity has been evaluated and correlated with liposome leakage. A transition from cell growth enhancement to cell death has been observed (C*∼500 nM, corresponding to 50-150% of an equivalent close packed monolayer for G2-G6 dendrimers, respectively) and is shown to correlate with the transition from cell membrane surface adhesion to internalization and nucleus interaction. Cytotoxicity also correlates qualitatively, but not quantitatively, with liposome leakage. A dendrimer concentration (500-700 nM) and surface coverage window for effective dendrimer internalization with low cytotoxicity was identified. These findings provide useful information for the successful design of dendrimers as drug or gene delivery vehicles. Acknowledgment. The authors would like to acknowledge funding from Starpharma Holdings Pty. Ltd. and Australian Research Council (ARC) Linkage grant scheme (LP0455268). S.P. acknowledges the ARC for an Australian Postgraduate Awards (Industry) scholarship. S.P. also acknowledges Dr. Ghafar Sarvestani for his guidance, training, and help with confocal microscopy. Ms. Renee Schulz, Dr. Paul Nielsen, and Dr. Raman Sharma are thanked for their training, support, and guidance in the cell biology studies.

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