Hydrophobic myristic acid modified PAMAM dendrimers augment the delivery of tamoxifen to breast cancer cells

Ishita Mataia and P. Gopinath*ab
aNanobiotechnology Laboratory, Centre for Nanotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand-247667, India. E-mail: pgopifnt@iitr.ernet.in; genegopi@gmail.com
bDepartment of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand-247667, India. Fax: +91-1332-273560; Tel: +91-1332-285650

Received 26th January 2016 , Accepted 26th February 2016

First published on 29th February 2016


Abstract

In the present study, cationic generation 5 polyamido amine (G5 PAMAM) dendrimers were hydrophobically modified by grafting the surface with lipid-like myristic acid (My) tails to augment their potential as a drug delivery vector in vitro. Nuclear magnetic resonance (1H NMR) measurements confirmed the presence of myristic acid tails at the dendrimer periphery (My-g-G5). Tamoxifen (TAM) an estrogen agonist, was entrapped in the My-g-G5 domains to impart them with anticancer properties. Transmission electron microscopy (TEM) observations indicate these My-g-G5/TAM complexes to be around 6–8 nm in size. Further, in vitro drug release studies ascertained the ability of My-g-G5/TAM complexes to release TAM in a sustained fashion under acidic conditions (pH 5.5). Cellular uptake studies revealed lysosomes as the target organelles of these nanocomplexes. MTT assay suggested good cell viability of My-g-G5 dendrimers and strong inhibitory effects of My-g-G5/TAM complexes in MCF-7 (human breast adenocarcinoma, estrogen receptor (ER) positive) cells. Dual fluorescence staining, reactive oxygen species (ROS) generation, cell cycle analysis, field emission scanning electron microscopy (FE-SEM), change in mitochondrial membrane potential (MMP, ΔΨ) and gene expression studies revealed the apoptosis-inducing ability of My-g-G5/TAM in MCF-7 cells. Based on our findings, we present these hydrophobically modified G5 PAMAM dendrimers as prospective nanocarriers for TAM delivery for anticancer applications.


1. Introduction

Polyamido amine (PAMAM) dendrimers with diverse functionalities have aroused the interest of researchers for basic understanding of their structure and function for various biological applications.1,2 These synthetic macromolecules are predominantly multivalent and offer wide flexibility for surface modification.3 Such special characteristics of PAMAM dendrimers make them a prospective candidate for biomedical applications. However, toxicity concerns related to PAMAM dendrimers bearing abundant positive charges hinders their applicability at clinical levels.3,4 Several modifications of PAMAM dendrimers have been attempted so far such as surface PEGylation,5,6 acetylation7,8 etc. to reduce their overall positive charge and hence the cytotoxicity. In our previous work, we have demonstrated that partial surface acetylated G5 PAMAM dendrimers exhibited reduced toxicity and could form hybrids with imaging agents like carbon dots (CQDs) to deliver anticancer drug epirubicin (EPI) to monitor drug delivery and response simultaneously.8

Also, addition of lipid components can significantly alter the physicochemical and biological properties of polycations for various biological and medical applications.9 Functionalization of cationic PAMAM dendrimers with hydrophobic chains were shown to have significantly improved transfection properties.10 For instance, G5 PAMAM dendrimers modified with lauric, myristic and palmitic fatty acids exhibited enhanced DNA binding ability and transfection efficiency to mesenchymal stem cells (MSCs) than unmodified dendrimers. Takahashi and co-workers synthesized low generation PAMAM G3 dendrons with two dodecyl chains and estimated their transfection efficiency as a function of dendron generation in CV1 cells.11 Kono et al. prepared G4 PAMAM dendrimers with hydrophobic amino acid residues (phenylalanine or leucine) at their chain ends.12 Joester et al. generated a series of amphiphilic dendrimers with diphenylethyne core by self-assembly as transfection agents.13 Additionally, lauroyl groups conjugated to low generation cationic PAMAM dendrimers reduced their cytotoxicity and increased permeability through Caco-2 cell monolayers.14 Overall, these studies have suggested improved performance of hydrophobically modified PAMAM dendrimers in biological systems than the unmodified ones.

Myristic acid (tetradecanoic acid, 14 carbons, My) is a saturated fatty acid with no toxicity reported till date.15,16 Such medium chain fatty acids are absorption enhancers and assist in the intracellular trafficking of proteins.14,17 Myristoyl attached polyarginine peptides are known to effectively penetrate the cytoplasmic and intracellular membranes.18 Also, myristic acid has been shown to enhance the gene delivery efficiency and brain targeting ability of polycations polyethylenimine (PEI25k).19,20

Herein, the main emphasis is to modify the surface of cationic PAMAM dendrimers by randomly grafting lipid-like myristic acid chains. Such modified dendritic entities comprise of backbone of PAMAM dendrimer with both amine groups and myristic acid tails at the periphery. These incorporated hydrophobic alkyl segments can act synergistically with the cationic core facilitating their interactions with cell membranes to effectively transport and deliver the therapeutic cargo.21 Also, such hydrophobic dendrimers with shielded positive charges are expected to have diminished cytotoxicity than unmodified dendrimers.14 Under physiological conditions, these myristic acid grafted dendrimers can enhance the solubility and bioavailability of poorly soluble/insoluble drugs. This can be attributed to the increased internal space in dendritic architecture after attaching lipid-like chains.

For this study, medium generation G5 PAMAM dendrimers were chosen owing their higher amine content (>G4) and lower toxicity (<G6) than other available cationic PAMAM dendrimers.22 Thereafter, we conjugated the surface amine groups of PAMAM dendrimers with the carboxylic groups of myristic acid using carbodiimide chemistry in a stoichiometric manner. Such modified PAMAM dendrimers with hydrophobic tails associate the advantages of cationic dendrimers and lipids to interact with cell membranes. Furthermore, we evaluated the ability of myristic acid grafted PAMAM dendrimers (My-g-G5) as drug delivery vectors. Tamoxifen (TAM) an estrogen agonist, is a U.S. FDA approved nonsteroidal drug for treatment and prevention of breast cancer. It binds to the estrogen hormone receptor (ER) expressed in breast cancer cells thereby blocking the binding site of estrogen.23,24 This eventually inhibits the tumor progression and reduces the risks of cancer recurrence.25 Owing to these, we chose TAM as a model drug for encapsulation within the interiors of My-g-G5 dendrimers. Various biological assays were performed to interpret the efficacy of My-g-G5 dendrimers to ferry TAM to inhibit and kill MCF-7 cells (human breast adenocarcinoma, ER positive) in vitro.

2. Experimental section

2.1. Materials

Amine terminated generation 5 polyamido (amine) dendrimers (G5 PAMAM) with ethylenediamine (EDA) core (Mw 28[thin space (1/6-em)]824.81 g mol−1) were purchased from Sigma-Aldrich, USA and subjected to filtration under reduced pressure to remove methanol content. Myristic acid (CH3(CH2)12COOH) was procured from SRL, India. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and tamoxifen (TAM, ≥99%) were bought from Sigma-Aldrich, USA. For cell culture experiments, 1,2-benzopyrone, 1-benzopyran-2-one, 2H-chromen-2-one (coumarin-6), 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyltetrazolium bromide (MTT), 2-(6-amino-3-imino-3H-xanthen-9-yl)benzoic acid methyl ester (rhodamine 123), 2′,7′-dichlorofluorescin diacetate (DCFDA), and 3,8-diamino-5-[3-(diethylmethylammonio)propyl]-6-phenylphenanthridinium diiodide (PI) were acquired from Sigma-Aldrich, USA. Acridine orange (AO) and ethidium bromide (EtBr) were obtained from Sigma-Aldrich, USA and SRL, India respectively. Cell staining dyes, Hoechst 33342 and LysoTracker red were bought from Life Technologies, USA. Dulbecco's minimum essential medium (DMEM) and phosphate buffer saline (DPBS; Ca2+ and Mg2+ free) powders were purchased from Sigma-Aldrich, USA and were reconstituted with ultrapure water (18 MΩ cm) before use.

2.2. Synthesis of myristic acid grafted G5 PAMAM (My-g-G5) dendrimers

The myristic acid chains were introduced on the periphery of G5 PAMAM dendrimers through EDC/NHS chemistry.26 Before PAMAM modification, myristic acid (0.043 mmol, 10 mg) was dissolved in 3 mL DMSO under N2 atmosphere. Then, EDC (0.043 mmol, 8.3 mg) and NHS (0.043 mmol, 5 mg) powders were added at once to myristic acid–DMSO solution. The solution was thereafter magnetically stirred for 12 h astray from light to activate the terminal carboxyl groups (–COOH) of myristic acid. Meanwhile, G5 PAMAM dendrimers (0.7 μmol, 20 mg) were separately dissolved in 3 mL DMSO in amber colored glass vials. After 12 h, the activated myristic acid solution was added drop-wise to the G5 PAMAM–DMSO solution under N2 atmosphere. The mixture solution was left undisturbed for 24 h at room temperature. Thereafter, the myristic acid–G5 PAMAM solution was dialyzed against PBS (2 L) for 1 day followed by water (2 L) for 3 days. The dialyzed solution was then freeze-dried to obtain pale-yellow colored My-g-G5 dendrimers. The samples were stored in powdered form at −20 °C until use.

2.3. My-g-G5/TAM complexation

Firstly, My-g-G5 dendrimers (10 mg) were dissolved in 1.5 mL double distilled water through high-speed vortexing. TAM with 10 molar equivalents of My-g-G5 dendrimers (1.28 mg) was dissolved separately in 300 μL methanol and added very slowly to the My-g-G5 aqueous solution.8 The mixture solution was kept uncovered under magnetic stirring to evaporate methanol for 24 h at room temperature. Then, My-g-G5/TAM solution was passed through nylon filter (pore size: 0.22 μm) to remove precipitates related to non-complexed free TAM. The concentration of TAM bound to My-g-G5 PAMAM dendrimers was estimated spectrophotometrically at 278 nm. An absorbance versus concentration calibration curve was generated using different concentrations of TAM. For data fitting, the least-squares approach was used (the regression equation at 278 nm was y = 0.035x − 0.068, R2 = 0.994). Similar procedure was adopted to complex TAM within the pristine G5 PAMAM dendrimers. All the samples were stored at −20 °C in dark.

For tracking the in vitro cellular uptake of My-g-G5 PAMAM dendrimers, a fluorescent dye coumarin-6 was complexed using the above procedure instead of TAM. The incorporated dye in My-g-G5 dendrimers can serve as an efficient and sensitive probe to mimic their intracellular uptake and localization.

2.4. Influence of pH on release of TAM from My-g-G5 PAMAM dendrimers

The release of TAM from My-g-G5 dendrimers was monitored in sodium acetate buffer (pH 5.5) and PBS (pH 7.4) using a Slide-A-Lyzer™ MINI dialysis device (10 kDa MWCO). Briefly, 1 mL of My-g-G5/TAM solution was added to the dialysis tube and the outer compartment was filled with 45 mL of buffer. The dialysis devices were mildly agitated at 100 rpm at 37 °C. At scheduled time intervals of 3, 6, 9, 12, 24, 36 and 48 h, 500 μL of buffer was withdrawn from the outer medium and replaced with equal volume of fresh buffer. The cumulative TAM in the medium was quantitated from its dose–absorption curve by measuring its absorbance at 278 nm. The % release was calculated as follows:
% TAM release = [(conc. of TAMaliquot)/initial TAM conc.] × 100

2.5. Characterization techniques

For nuclear magnetic resonance (NMR) measurements, samples were dissolved in DMSO-d6 solvent in millimolar concentrations and 1H NMR spectra were recorded at RT, using a Bruker 500 MHz NMR spectrometer. Zeta potential measurements were carried out for the modified dendrimers (1 mg mL−1 concentration) in different medium using Malvern Nano ZS 90, Zetasizer operating at 25 °C. UV-visible spectroscopic experiments were conducted using Lasany LI-2800, UV-vis spectrophotometer. Transmission electron microscopic (TEM) analysis was performed with an FEI TECHNAI G2 analytical electron microscope operating at 200 kV. 20 μL of My-g-G5/TAM sample was dropped onto carbon coated copper grid and left for 10 min. Excess sample was removed carefully and then the grid was negatively stained with 2% phosphotungstic acid (PTA), air dried and then viewed under TEM.

2.6. Cell culture

Cell lines MCF-7 (human breast adenocarcinoma, ER+) and NIH 3T3 (mouse embryonic fibroblast, ER−) purchased from the National Centre for Cell Sciences (NCCS) Pune, India were cultured in DMEM medium supplemented with 10% (v/v) FBS and 1% (v/v) penicillin–streptomycin. The cells were maintained in 5% CO2 incubator @ 37 °C and subcultured after 60–70% confluency with 0.25% trypsin–EDTA.

2.7. MTT assay

The cellular viability was quantitated by measuring the percentage of live cells when exposed to various dendrimer formulations. Briefly, MCF-7 and NIH 3T3 cells at a density of 10[thin space (1/6-em)]000 cells per well (100 μL total volume per well) were seeded in 96-well plates for 24 h. Then, the cell medium was removed and fresh DMEM mixed with G5, My-g-G5, G5/TAM, My-g-G5/TAM, and free TAM (with equivalent concentrations) were added to cells for 48 h of exposure. Healthy MCF-7/NIH 3T3 cells without treatment were considered as negative control. Following treatment, the medium was aspirated and PBS wash was given to get rid of residual particles. 10 μL of MTT dye (5 mg mL−1) was added per well and incubated for 4 h in 5% CO2 at 37 °C for formation of purple formazon crystals. Thereafter, 100 μL DMSO was added per well to dissolve the formazon product and the color intensity was estimated by measuring absorbance at 570 nm using microplate reader (Cytation3, Biotek). All the readings were taken in triplicate and the cell viability (%) was calculated as:
Cell viability (%) = [A570 treated cells/A570 control cells] × 100

2.8. Cellular uptake studies

The uptake of My-g-G5/C6 complexes by MCF-7 cells was examined qualitatively using fluorescence microscopy and quantitatively through flow cytometry. MCF-7 cells (2 × 105 cells) were cultured in 6-well plates and incubated with My-g-G5/C6 (0.75 and 1 μM) complexes for 6 h. Unexposed MCF-7 cells were used as internal control. Subsequently, cells were observed under fluorescence microscope (EVOS® FL Color, AMEFC 4300) in the GFP filter. For quantitating green positive cells (%), the cells were washed with PBS, trypsinized carefully and harvested by centrifugation at 600 g for 6 min at 4 °C. The attained cell pellets were then dispersed in 200 μL PBS and analyzed for green fluorescent signals in Ch. 02 (505–560 nm) by flow cytometer (Amnis Flowsight). A total of 10[thin space (1/6-em)]000 events were acquired per sample and analyzed using Amnis Ideas software.

The cellular location of My-g-G5/C6 complexes was also determined in a time dependent manner. For this, MCF-7 cells (2 × 105 cells) were seeded and cultured in 35 cm plates and exposed to My-g-G5/C6 (0.75 μM) for 1, 2, 4 and 6 h. Thereafter, cells were given a PBS wash followed by staining with 2 μL of Hoechst 33342 dye (10 mg mL−1) and 2 μL of lysotracker red (100 μM) for 10–15 min at 37 °C. Images were then recorded under various filters (transmitted, DAPI (λex 360 nm, λem 447 nm), GFP (λex 470 nm, λem 525 nm), and RFP (λex 530 nm, λem 593 nm)) of an inverted fluorescence microscope (EVOS® FL Color, AMEFC 4300).

2.9. Acridine orange/ethidium bromide (AO/EB) staining

MCF-7 cells treated with G5/TAM and My-g-G5/TAM samples were subjected to AO/EB dual dye to perceive the occurrence of apoptosis. For this, 2 × 105 MCF-7 cells were seeded in 6-well cell culture plates and treated with desired concentrations of G5/TAM and My-g-G5/TAM (0.75 and 1 μM), respectively for 24 h. After treatment, medium was removed and cells were washed with PBS. Cells were then stained with AO/EB dye (10 μg mL−1 working conc.) for 15–20 min at 37 °C. Images were then captured under different filters using an inverted fluorescent microscope (EVOS® FL Color, AMEFC 4300).

2.10. Determination of mitochondrial membrane potential (MMP)

Alterations in MMP (ΔΨ) in MCF-7 cells was evaluated by rhodamine 123 staining. MCF-7 cells (2 × 105 cells per well) were seeded in 6-well plates for overnight attachment. Cells were then exposed to G5/TAM and My-g-G5/TAM (0.75 and 1 μM) complexes for 24 h. Untreated MCF-7 cells were also kept as control. After exposure cells were washed twice with PBS, trypsinized and harvested by centrifugation at 600 g for 6 min at 4 °C. The cell pellet was then resuspended with 200 μL PBS containing rhodamine 123 (5 μg mL−1) and kept for 30 min at 37 °C. The fluorescence intensities of rhodamine 123 in cells were quantitated by flow cytometry (Amnis Flowsight). For fluorescent microscopy, in parallel treated MCF-7 cells (seeded in another plate) were washed with PBS and stained with equivalent conc. of rhodamine 123 (5 μg mL−1) for 30 min. Images were captured for red fluorescent signals under RFP filter (λex 530 nm, λem 593 nm) at 40× magnification of fluorescent microscope (EVOS® FL Color, AMEFC 4300).

2.11. Measurement of reactive oxygen species (ROS) generation

The intracellular ROS generation when exposed to My-g-G5/TAM complexes was monitored by DCFH-DA staining. Basically, DCFH-DA is a non-fluorescent cell permeable dye, which is hydrolysed to DCFH and thereby oxidized to fluorescent DCF in presence of intracellular ROS. For this, MCF-7 cells (2 × 105 cells) were plated in 6-well plate and nursed for 24 h. After that, cell medium diluted with My-g-G5/TAM (0.75 and 1 μM) complexes were added to the attached cells for 24 h. Next, cells were washed, trypsinized and harvested by centrifugation at 600 g for 6 min at 4 °C. Obtained cell pellets were then redispersed in PBS with 20 μM DCFH-DA and kept for 10 min incubation at 37 °C in dark. The samples were then analyzed for DCF fluorescence by flow cytometry (Amnis Flowsight). A total of 10[thin space (1/6-em)]000 events were acquired per sample for statistical analysis. Green fluorescent images were also captured using a fluorescent microscope (EVOS® FL Color, AMEFC 4300) in the GFP channel (λex 470 nm, λem 525 nm) at 40× magnification.

2.12. Cell cycle analysis

For cell cycle analysis, cells were stained with propidium iodide (PI) and subsequently analyzed by flow cytometry. MCF-7 cells (2 × 105 cells) were cultured in 6-well plate and exposed to My-g-G5/TAM (0.75 and 1 μM) samples for 24 h. Following treatment, cells were washed with PBS, trypsinized and centrifuged at 200 g for 5 min at 4 °C. The obtained cell pellet were then fixed with 70% ethanol in ice for 15 min. The fixed cells were then resuspended in PI staining solution (50 μg mL−1 PI, 1 mg mL−1 RNase A, and 0.05% triton X-100) and incubated for 45 min at 37 °C in dark. After incubation, the cells were immediately analyzed using flow cytometer (Amnis Flowsight).

2.13. Gene expression studies

The differential signalling pattern of apoptosis related genes was interpreted by semi-quantitative RT-PCR. MCF-7 cells (2 × 105 cells) were grown in 35 mm cell culture dishes and exposed to DMEM containing My-g-G5/TAM (0.75 and 1 μM) complexes. After treatment, medium was removed, a mild PBS wash was given and cells were lysed with Tri reagent (Sigma Aldrich, USA). Total RNA was isolated using the manufacturer's protocol and cDNA was generated by reverse transcription (Super Script II Reverse Transcriptase, Life Technologies India) of 1 μg of extracted RNA. The PCR reaction was set in a RT-PCR (Applied Biosystems) with the generated cDNA with the steps which include: initial denaturation (94 °C for 3 min) followed by PCR cycle of denaturation (94 °C for 30 s), annealing (60 °C for 30 s), extension (72 °C for 1 min), and a final extension (72 °C for 10 min). The PCR products were finally electrophoresed in a 1% agarose gel for 50 min at 60 V.

2.14. Cell morphology analysis

The cell morphology after My-g-G5/TAM exposure was investigated through FE-SEM analysis. Firstly, MCF-7 cells (2 × 105 cells) were seeded on glass coverslips in 35 mm plates and treated with My-g-G5 PAMAM/TAM (0.75 and 1 μM) complexes for 24 h. After that, cells were washed with PBS and fixed with 2% glutaraldehyde solution for 5–10 min followed by ethanol gradient fixation (20%, 40%, 60%, and 80%). Cells were then gold sputtered and viewed under FE-SEM (Ultra plus-Carl Zeiss) operating at 5 kV.

3. Results and discussion

3.1. Preparation and characterization of My-g-G5/TAM complexes

The main objective of this study was to combine the magnificent properties of PAMAM dendrimers and lipophilic moieties to realize a nanoscale delivery system with the desired biological features. With this idea, myristic acid (a fatty acid) chains chiefly hydrophobic in nature were linked to the periphery of G5 PAMAM dendrimers. EDC/NHS chemistry was employed to form amide linkage (–CONH–) between the terminal carboxyl group (–COOH) of myristate and amines (–NH2) of dendrimers.26 After successful incorporation of myristic acid chains on the dendrimers, we evaluated My-g-G5 system as a carrier for encapsulating water insoluble TAM drug. G5 PAMAM dendrimers with hydrophobic cavities can stabilize and increase the water solubility of TAM.27 This is primarily important to achieve the desired therapeutic benefits by enhancing the drug circulation times. The entire synthesis procedure of preparation of My-g-G5/TAM complexes has been depicted in Scheme 1. The as-synthesized My-g-G5/TAM samples were stored at −20 °C in powdered form and were found to be stable for several months without any loss in TAM activity.
image file: c6ra02391f-s1.tif
Scheme 1 Schematic representation of preparation of My-g-G5/TAM complexes. The entire synthesis scheme involves multiple steps which include: (1) EDC/NHS activation of myristic acid, (2) chemical reaction of activated myristic acid and G5 PAMAM dendrimers and (3) complexation of TAM in the internal spaces of My-g-G5 dendrimers to form My-g-G5/TAM complexes.

These My-g-G5 dendrimers were first characterized by 1H NMR measurements to ascertain the extent of functionalization. Fig. 1 represents the 1H NMR spectra of My, My-g-G5 and G5 dendrimers in DMSO-d6 solvent. As seen, the characteristic proton peaks of myristic acid at 0.8 and 1.25 ppm of –CH3 and –CH2 groups were present in My-g-G5 along with its other main peaks (2.2–3.4 ppm). Based on integrals of proton peaks at 0.8 ppm (of myristic acid) and 2.3 ppm (of G5), ∼29 myristic acid chains were present per dendrimer. Hence from 1H NMR findings, the functionalization of dendrimer exterior with myristic acid chains was confirmed. Subsequently, we evaluated the potential stability of My-g-G5 dendrimers by performing zeta potential measurements in acetate buffer, PBS and DMEM medium, respectively. Fig. S1A, shows the variation in the zeta potential profile of My-g-G5 dendrimers with change in medium. When added to acetate buffer (pH 5.5), the zeta potential of My-g-G5 was 41.3 ± 1.18 mV suggesting their excellent stability in acidic conditions. While, the zeta potential value declined to 15.7 ± 1.11 mV when added to PBS (pH 7.4). A flip in the charge of My-g-G5 dendrimer solution was observed when added to DMEM medium, it was −5.12 ± 0.426 mV suggesting slight reduction in dendrimer stability owing to its obvious interaction with the medium constituents. However, with visual examination the My-g-G5 dendrimers (at 1 mg mL−1 concentration) displayed good solubility in acetate buffer, PBS and DMEM medium and no aggregation was observed (Fig. S1B). This suggests that even after attachment of some My chains on the dendrimer periphery, they retained their solubility properties. Thereafter complexation with TAM, we characterized the formed My-g-G5/TAM complexes by UV-visible spectroscopy. The absorption spectra of free TAM in methanol and blank My-g-G5 dendrimers were also recorded for comparison. As shown in Fig. S2, TAM exhibits two characteristic peaks at 238 and 278 nm. After encapsulation, My-g-G5/TAM complexes exhibits a prominent peak ∼278 nm when compared to blank My-g-G5 dendrimers. Presence of peak enhancement at 278 nm in My-g-G5/TAM complexes indicate successful encapsulation of TAM in My-g-G5 dendrimers. Estimated from UV-vis analysis and dose dependent TAM curve, the encapsulation efficiency was calculated to be ∼55.6%. Next, we visualized the morphology and size of My-g-G5/TAM complexes under TEM. Fig. 2 shows the TEM micrographs of My-g-G5/TAM complexes after negative staining with PTA. As evident from the results, My-g-G5/TAM complexes were uniform in distribution with near spherical morphology. The average size of these complexes were in range of 6–8 nm, larger than PAMAM G5 size (5.4 nm). This size increment is indicative of presence of myristic acid segments at the dendrimer surface.


image file: c6ra02391f-f1.tif
Fig. 1 1H NMR spectra of different samples at RT. The blue highlighted portions reflect similar regions.

image file: c6ra02391f-f2.tif
Fig. 2 High resolution TEM image of My-g-G5/TAM complexes after negative staining (scale bar: 50 nm).

3.2. In vitro TAM release studies

To monitor the ability of My-g-G5/TAM complexes to release the therapeutic payload, we exposed My-g-G5/TAM complexes to buffers with different pH and monitored the release of TAM with time. Fig. 3 depicts the cumulative release profile of TAM as a function of time at pH 5.5 and 7.4, respectively. As observed, My-g-G5 dendrimers were proficient to release TAM over a span of time with more release under acidic conditions (tumor microenvironment) than basic environments (physiological environment). For instance, after 48 h ∼67% TAM was released at pH 5.5 while only ∼28% drug was released at pH 7.4. The fast drug release at acidic pH can be owed to the protonation of the interior amines of My-g-G5 dendrimers (pKa ∼ 6.3).28 After in vitro testing, we estimated the anticancer properties of My-g-G5/TAM complexes under cellular conditions.
image file: c6ra02391f-f3.tif
Fig. 3 Cumulative release profile of TAM from My-g-G5/TAM complexes as a function of time at pH 5.5 (red) and 7.4 (black), respectively.

3.3. Effect of My-g-G5/TAM complexes on cell viability

The cytotoxic effects of as as-synthesized My-g-G5/TAM complexes were then tested in vitro in MCF-7 and NIH 3T3 cells by MTT assay. As shown in Fig. 4A, MCF-7 cells incubated with G5 and My-g-G5 were able to maintain a good cell viability (∼80%) at the tested drug concentrations (0.25–0.75 μM). A slight decrease in cell viability with My-g-G5 at 1 μM, may be due its enhanced interaction with the cells. Unmodified G5/TAM complexes didn't show any signs of cell inhibition and death at all the tested dosages. Contrarily, the My-g-G5/TAM complexes were found to significantly alter the MCF-7 survival and induce cell death. With increase in drug concentration from 0.25–1 μM, the cell viability drastically reduced from 83.8% to 27.1%. Since, within this concentration regime My-g-G5 dendrimers did not display much cytotoxicity, the plausible explanation to the reduced cell viability could be due to the action of the entrapped TAM molecules. Moreover, this observed cell viability was similar to that observed with free TAM (equivalent concentrations).29 This suggests that My-g-G5 dendrimers were able to preserve the TAM activity in cellular environment to inhibit the ER positive cancer growth. Moreover, such significant difference in the cell viabilities of G5/TAM and My-g-G5/TAM complexes can be designated to the surface modification of dendrimers with lipophilic tails which enhanced the TAM stability and interaction with the breast cancer cell membranes.
image file: c6ra02391f-f4.tif
Fig. 4 Viability of (A) MCF-7 and (B) NIH 3T3 cells after treatment with different concentrations of G5, My-g-G5, G5/TAM, My-g-G5/TAM and free TAM after 48 h calculated from MTT assay. The values are expressed as mean ± standard error mean (SEM) (n = 3). Two-way ANOVA with Tukey's multiple comparisons test was used to determine statistical difference between the group means (**p < 0.005, ***p < 0.001, ns: non-significant).

We also evaluated the effects of My-g-G5/TAM complexes on normal NIH 3T3 cells. As expected, under similar treatment conditions the cells incubated with G5 PAMAM dendrimers showed some cytotoxic effects and reduced the cell viability to nearly ∼60% (Fig. 4B). However, cells in presence of My-g-G5 or My-g-G5/TAM complexes exhibited an appreciable cell viability (>80%). This recommends that introduction of myristoyl moieties on dendrimer surface reduces its cytotoxicity. Moreover, free TAM was found to inhibit the cell growth at high concentrations. These results conclude that at similar dosage, the inhibitory effects of My-g-G5/TAM complexes were more pronounced in MCF-7 than NIH 3T3 cells. Hence, My-g-G5 dendrimers with hydrophobic corona are a more suitable alternative to unmodified G5 PAMAM dendrimers for TAM delivery and induction of breast cancer cell death in vitro.

3.4. Investigation of cellular uptake

An important parameter which affects the therapeutic outcome of most drugs is the effective cellular uptake of its carrier. Most nanocarriers tend to deliver their drug cargos by passively targeting and accumulating within the cancer cells.30 Hence it is imperative to track the intracellular distribution of chemotherapeutics after its uptake. Since TAM is a non-fluorescent drug, we prepared coumarin-6 loaded My-g-G5 dendrimers to monitor and quantitate the cellular uptake. MCF-7 cells were incubated with different concentrations of My-g-G5/C6 complexes for 6 h and viewed after fluorescent microscopic imaging. As evident from Fig. 5 the green fluorescence from MCF-7 cells increased in a dose-dependent manner. We then quantitated the intracellular fluorescence intensity using flow cytometry. As expected, with shift in the concentration of My-g-G5/C6 from 0.75–1 μM, the percentage of green positive cells indicating uptake of My-g-G5/C6 complexes increased from 66.6% to 84.9% (Fig. 5(b2) and (c2)). The obtained results indicate fruitful internalization and accumulation of My-g-G5 dendrimers in cancer cells.
image file: c6ra02391f-f5.tif
Fig. 5 Fluorescence microscopic images of MCF-7 cells incubated with different concentrations of My-g-G5/C6 complexes (a1–c1) and the corresponding quantitation of cellular uptake by flow cytometry (a2–c2) (scale bar: 100 μm).

Next we tried to track the localization of My-g-G5/C6 complexes in cancer cells using Hoechst 33342 as a marker for nuclei and lysotracker red for fluorescence imaging of lysosomes. As evident from Fig. 6 the green fluorescent signals increased with time and were distributed in the cytoplasm of cells. However, we did not observe any overlay of green and blue fluorescence which suggests that nuclei were not the cellular targets of My-g-G5/C6 complexes. On the contrary, the red fluorescence from lysosomes colocalized with the green fluorescence which eventually increased with time. These observations clearly demonstrate lysosomes as the residing compartment of My-g-G5/C6 complexes. It is important to mention that some My-g-G5/C6 managed to escape the lysosomes and were released in the cell cytoplasm. These results corroborate with the previously reported findings whereby PAMAM dendrimers target lysosomes after endocytosis and are released by the “proton-sponge effect”.31,32


image file: c6ra02391f-f6.tif
Fig. 6 Fluorescence microscopic images revealing time dependent cellular uptake of My-g-G5/C6 complexes (0.75 μM) in MCF-7 cells under different filters (scale bar: 100 μm).

3.5. Induction of apoptotic cell death by My-g-G5/TAM complexes

We then tried to investigate the mode of death (viz. apoptosis or necrosis) after treatment with My-g-G5/TAM complexes using AO/EB dual dye. Fig. 7 shows that MCF-7 cells treated with 0.75 μM G5/TAM complexes (Fig. 7b) had uniform morphology and displayed green fluorescence similar to untreated ones (Fig. 7a). A slight onset of apoptosis was viewed based on yellow-green fluorescence from cells treated with 1 μM G5/TAM (Fig. 7c). However, upon treatment with My-g-G5/TAM complexes, both yellow-green and orange-red stained nuclei suggest early and late apoptosis, respectively (Fig. 7d). The proportion of cells in late apoptotic stage increased when treated with 1 μM My-g-G5/TAM complexes (Fig. 7e). Thus, AO/EB staining signifies manifestation of apoptosis in My-g-G5/TAM complexes treated MCF-7 cells.
image file: c6ra02391f-f7.tif
Fig. 7 AO/EB stained images of (a) untreated, (b and c) G5/TAM and (d and e) My-g-G5/TAM (0.75 and 1 μM) treated MCF-7 cells. Yellow arrows indicate cells undergoing early apoptosis and white arrows point towards the late apoptosis stages (scale bar: 100 μm).

3.6. Fluctuation in mitochondrial membrane potential (MMP)

Mitochondria is one of the key cell organelle affected during the apoptotic pathway of cell death. Change in the mitochondrial membrane permeability with a loss in MMP (ΔΨ) is an indicator of early apoptotic events.33 To explore whether the apoptosis induced by My-g-G5/TAM complexes involved the loss of mitochondrial integrity and hence ΔΨ, a cationic dye rhodamine 123 was used. Rhodamine 123 can rapidly diffuse inside the mitochondrial interior and can reflect the changes in MMP.34 MCF-7 cells exposed to different concentrations of G5/TAM and My-g-G5/TAM complexes were stained with rhodamine 123 and its fluorescence intensity was observed. In MCF-7 cells treated with G5/TAM (0.75 and 1 μM) the intensity of red fluorescence was identical to the untreated cells (Fig. 8(a1)–(c1)) i.e., no loss in MMP was observed. In comparison, cells treated with My-g-G5/TAM (0.75 and 1 μM) displayed a significant decline in the red fluorescent signal, suggesting reduced uptake of rhodamine 123 or loss of ΔΨ (Fig. 8(d1) and (e1)). To support this observation, the percentage of cells with rhodamine 123 uptake were quantitated by flow cytometry. As evident, the percentage of red positive cells reduced from 61.1% (without treatment) to 51.1% and 37.3% after My-g-G5/TAM (0.75 and 1 μM) treatment, respectively (Fig. 8(a2), (d2) and (e2)). Whereas, the population of positive cells after G5/TAM exposure remained unaltered (70.4% and 64.5%). These results reveal a remarkable decline in MMP confirming induction of apoptosis in MCF-7 cells after My-g-G5/TAM treatment.
image file: c6ra02391f-f8.tif
Fig. 8 Fluorescence microscopic images of rhodamine 123 stained (a1) untreated, (b1 and c1) G5/TAM and (d1 and e1) My-g-G5/TAM (0.75 and 1 μM) treated MCF-7 cells (scale bar: 100 μm). The other panel (a2–e2) depicts the corresponding flow quantitation. Decline in the fluorescence intensity of rhodamine 123 represents loss in mitochondrial membrane potential (MMP).

3.7. My-g-G5/TAM treatment triggers ROS production

An increase in the cellular ROS species or oxidative stress is known to elicit apoptotic cell death. The intracellular ROS production was evaluated in MCF-7 cells after exposure to different concentrations of My-g-G5/TAM (0.75 and 1 μM) complexes by DCFH-DA staining. The fluorescence images reveal an intensification in the DCF green fluorescence after treatment compared to untreated cells (Fig. 9(a1)–(c1)). When quantified, the percentage of cells with ROS increased from 11.7% (in untreated cells) to 29.6% after My-g-G5/TAM (0.75 μM) treatment (Fig. 9(a2) and (b2)). However, at higher concentration of My-g-G5/TAM (1 μM), the percentage of green positive cells reduced to 16.7%, owing to their increased killing and leakage of ROS.35
image file: c6ra02391f-f9.tif
Fig. 9 Microscopic and flow cytometric analysis of (a1 and a2) untreated, My-g-G5/TAM treated MCF-7 cells with (b1 and b2) 0.75 μM and (c1 and c2) 1 μM, respectively for ROS generation after DCFH-DA staining (scale bar: 100 μm).

3.8. Effect of My-g-G5/TAM treatment on cell cycle and gene expression profile

To probe the effect of My-g-G5/TAM treatment in altering the cell cycle stages, PI staining was performed and cells were thereafter quantitated. As shown in Fig. 10A, a dose dependent increase in cells arrested at sub G0/G1 stage was observed with My-g-G5/TAM treatment. These cells in the sub G0/G1 phase with sub-diploid DNA content primarily constitutes the apoptotic population.8,36 The % apoptotic cells reached to around 12.1% and 22.9% after My-g-G5/TAM treatment (0.75 and 1 μM), respectively. Moreover, the ability of anti-estrogen TAM to modulate the expression of genes involved in mitochondrial mediated apoptotic pathway is well-documented.37,38 In this context, we monitored the differential expression of pro-apoptotic p53, bax, bad, caspase 3 and anti-apoptotic bcl-xl genes. Beta actin (a housekeeping gene) was kept as an internal control. Fig. 10B shows the RT-PCR analysis of these apoptotic genes after My-g-G5/TAM treatment. The obtained data clearly depicts an up-regulation in the pro-apoptotic gene expression in a dose dependent manner. On the other hand, with increase in treatment concentration a significant down-regulation was observed in bcl-xl expression levels. The expression of beta actin remained unchanged. The prominent trigger of pro-apoptotic p53, bax, bad and caspase 3 mRNA levels suggests instigation of gene signalling cascade in MCF-7 cells. For comparative gene expression analysis, we exposed MCF-7 cells to G5/TAM and My-g-G5/TAM complexes (with equivalent TAM concentrations, 0.75 μM) under similar conditions. We observed that the expression of pro-apoptotic genes in response to G5/TAM were comparable to the untreated cells (Fig. S3). On the other hand, the same genes were found to be up-regulated with My-g-G5/TAM exposure compared to G5/TAM. The deviation in regulation of gene profile of apoptosis related genes suggest superiority of My-g-G5 dendrimers over pristine G5 PAMAM dendrimers. These results signifies that My-g-G5/TAM complexes were effective enough to regulate the expression of genes directly involved in apoptotic pathway.
image file: c6ra02391f-f10.tif
Fig. 10 (A) Cell cycle and (B) semi quantitative RT-PCR analysis of MCF-7 cells treated with different concentrations of My-g-G5/TAM complexes. Multiple t tests using the Holm–Sidak method, was used to compute the statistical significance (*p < 0.05, **p < 0.005).

3.9. Morphological changes associated with My-g-G5/TAM treatment

We further analyzed the changes in cell morphology and appearance owing to apoptosis induction under FE-SEM. Fig. 11 depicts the typical FE-SEM micrographs of untreated and treated MCF-7 cells. Untreated MCF-7 cells appeared healthy and were well-attached to the surface (Fig. 11a). At lower dose of My-g-G5/TAM (0.75 μM), membrane constriction and shrinkage was seen and cells were loosely-adhered to the surface (Fig. 11b). Moreover at higher dose, the cells became round and typical morphological transformations related to apoptosis including membrane blebbing and appearance of apoptotic bodies were observed (Fig. 11c and d).39,40 Such findings are valuable while designing nanocarriers for achieving the desired therapeutic benefits for cancer inhibition and prevention. In totality, surface engineering of PAMAM dendrimers by randomly decorating myristoyl chains can be an intelligent strategy to design delivery vectors with improved benefits.
image file: c6ra02391f-f11.tif
Fig. 11 FE-SEM images of (a) untreated and (b and c) My-g-G5/TAM (0.75 and 1 μM) treated MCF-7 cells, respectively. (d) Magnified view of (c) depicting apoptotic morphology of cells.

4. Conclusion

In this work, we have explored the advantages of hydrophobically modified G5 PAMAM dendrimers for the delivery of anti-estrogen tamoxifen (TAM). Random grafting of lipid-like myristic acid chains on the dendrimer surface significantly enhanced the stability and solubility of TAM in polar conditions. Moreover, introduction of myristoyl groups improved the cellular uptake and decreased the cytotoxicity of PAMAM dendrimers. We have further elucidated the apoptosis inducing ability of My-g-G5/TAM complexes in MCF-7 (human breast cancer) cells by different cell based experiments. To conclude, we present these surface engineered PAMAM dendrimers as a unique nanoscale delivery system combining the advantages of both lipid based carriers and dendritic systems for anticancer therapeutics.

Acknowledgements

We give sincere thanks to the Science and Engineering Research Board (No. SR/FT/LS-57/2012) and Department of Biotechnology (No. BT/PR6804/GBD/27/486/2012), Government of India, for the financial support. IM is thankful to the Ministry of Human Resource Development, Government of India, for the fellowship. We sincerely acknowledge Institute Instrumentation Centre (IIC) at Indian Institute of Technology Roorkee for providing NMR, TEM and FE-SEM facilities.

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Footnote

Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra02391f

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