This content has been downloaded from IOPscience. Please scroll down to see the full text.

Download details:

IP Address: 193.40.12.10

This content was downloaded on 15/04/2015 at 08:56

Please note that terms and conditions apply.

A new technique for reversible permeabilization of live cells for intracellular delivery of

quantum dots

View the table of contents for this issue, or go to the journal homepage for more

2013 Nanotechnology 24 205101

(http://iopscience.iop.org/0957-4484/24/20/205101)

Home Search Collections Journals About Contact us My IOPscience

IOP PUBLISHING NANOTECHNOLOGY

Nanotechnology 24 (2013) 205101 (13pp) doi:10.1088/0957-4484/24/20/205101

A new technique for reversiblepermeabilization of live cells forintracellular delivery of quantum dots

Krishnakiran Medepalli1, Bruce W Alphenaar1, Robert S Keynton2 andPalaniappan Sethu2,3

1 Department of Electrical and Computer Engineering, Speed School of Engineering,University of Louisville, Louisville, KY 40208, USA2 Department of Bioengineering, Speed School of Engineering, University of Louisville, Louisville,KY 40208, USA

E-mail: p.sethu@louisville.edu

Received 26 January 2013, in final form 26 March 2013Published 19 April 2013Online at stacks.iop.org/Nano/24/205101

AbstractA major challenge with the use of quantum dots (QDs) for cellular imaging and biomoleculardelivery is the attainment of QDs freely dispersed inside the cells. Conventional methods suchas endocytosis, lipids based delivery and electroporation are associated with delivery of QDsin vesicles and/or as aggregates that are not monodispersed. In this study, we demonstrate anew technique for reversible permeabilization of cells to enable the introduction of freelydispersed QDs within the cytoplasm. Our approach combines osmosis driven fluid transportinto cells achieved by creating a hypotonic environment and reversible permeabilization usinglow concentrations of cell permeabilization agents like Saponin. Our results confirm thathighly efficient endocytosis-free intracellular delivery of QDs can be accomplished using thismethod. The best results were obtained when the cells were treated with 50 µg ml−1 Saponinin a hypotonic buffer at a 3:2 physiological buffer:DI water ratio for 5 min at 4 ◦C.

S Online supplementary data available from stacks.iop.org/Nano/24/205101/mmedia

(Some figures may appear in colour only in the online journal)

1. Introduction

Nanomaterials are comparable in size to various biomolecules(1–100 nm) and have unique properties such as enhancedelectrical conductivity, increased chemical reactivity, andnovel optical properties, making them attractive candidates forvarious biomedical applications [1–4]. The size and opticalproperties of nanomaterials have been exploited to developefficient tools for sub-cellular imaging and biomoleculardelivery [5]. Traditional bimolecular delivery methodsutilize plasmids, cationic polymers, lipids, and viruseswhich have inherent disadvantages such as degradation inphysiological solutions and the need for complex conjugation

3 Address for correspondence: University of Louisville, 2210 S. BrookStreet, SRB 357, Louisville, KY 40208, USA.

techniques [6–8]. Quantum dots (QDs) are nanometer sizedsemiconductor crystals typically between 2 and 6 nm indiameter. QDs have high chemical and biochemical stabilityand their small size and large surface area allow simultaneousconjugation with multiple biomolecules. QDs also haveseveral advantages over traditional fluorescent molecules(organic dyes) such as high resistance to photobleachingand tunable emission wavelengths based on the QD corediameter and composition, making them highly desirablefor different biomedical applications as imaging agents andbiomolecular delivery vehicles [9]. There is some concernregarding the cytotoxicity of QDs as they are made of heavymetal atoms like Cd, Hg, Pb and As. Several studies haveinvestigated the toxicity of QDs by studying the release ofCd+2 ions, as these can bind to sulfhydryl groups present inmany biomolecules like proteins and cause a decrease in the

10957-4484/13/205101+13$33.00 c© 2013 IOP Publishing Ltd Printed in the UK & the USA

Nanotechnology 24 (2013) 205101 K Medepalli et al

functionality of various sub-cellular organelles [10]. However,coating these QDs with ZnS shells virtually minimized thetoxicity, indicating that the shell protects the release of thecadmium [11]. More recently, green chemistry based andnon-cadmium based QDs have been synthesized for reducedtoxicity in biomedical applications [12].

Typically, the QDs are dispersed in aqueous buffersolutions and have been successfully functionalized withvarious biomolecules like DNA, proteins and antibodiesfor delivery applications [13]. However, a major challengeis the efficient intracellular delivery of monodispersedQDs–bioconjugates freely dispersed in the cytoplasm. Severalmethods have been developed for the delivery of QDsinto cells including both biochemical methods such asendocytosis, pinocytosis, lipids and physical methods such aselectroporation and micro/nanoinjection [5]. Thus far, therehave been demonstrations of receptor mediated endocytosisfor intracellular delivery; however the QDs remain trappedin the endocytic vesicles which are difficult to lyse,preventing their free diffusion within the cytoplasm [13, 14].Quantum dots complexed within transfection agents suchas cationic liposomes have been shown to be endocytosedand subsequently escape from the endosomes for freerelease into the cytoplasm [15]. Macropinocytosis has beenused for delivering macromolecules into cells, followingwhich osmotic lysis of pinocytic vesicles needs to beaccomplished [16]. Courty et al demonstrated the deliveryof QDs into live cells using a pinocytosis procedure [17]to detect intracellular movement of single molecular motors.Physical methods like electroporation have also been usedto deliver large quantities of quantum dots into cells [15].These methods though successful in delivery of QDs into thecellular cytoplasm, accomplish delivery of QDs as aggregatesthat are not monodisperse, limiting the utility of deliveredQDs. Endocytosis delivered QDs are trapped in the vesicles,preventing the labeling of other intracellular components suchas mitochondria or the nucleus. Delivery of QDs as aggregatesrestricts their subsequent trafficking such as translocationto the nucleus. Moreover, the biomolecule/antibody (tobe delivered) attached to the QDs remains sequestered inthe vesicles without it being free in the cytoplasm, thusmaking it unavailable for subsequent molecular recognitionand thereby limiting its function. Aggregation of QDs notonly affects their stability but also can result in quenchingof the fluorescence emission and thus a loss of opticalproperties. The only technique that has been shown toaccomplish uniform distribution of QDs into the cytosol ornucleus without endosomal trapping is microinjection [15,18]. However this technique is a laborious and low throughputprocess accomplished by injecting one or few cells, which is asignificant drawback for various applications [15, 18]. Hence,an efficient method to accomplish freely diffused QDs insidecells is needed for the labeling of cellular components as wellas intracellular delivery of biomolecules.

In this study, we demonstrate a new technique forreversible permeabilization of live cells for delivery of QDs.To minimize the effect of the endocytotic internalizationpathway of QDs, experiments were performed at 4 ◦C to

minimize the energy available for endocytosis [19]. Ourtechnique is based on creating a hypotonic environmentoutside the cells. Hypotonic exposure causes an influx of fluidinto the cell due to the osmotic pressure gradient, which canbe exploited to transport QDs along with the fluid into thecytoplasm of the cell. Our group and others have demonstratedthat osmosis based methods can be used to transportfluids [20–22] and other macromolecules into cells [16, 23,24]. To enhance the QDs’ delivery and dispersion in the cells,a cell permeabilization agent was used in conjunction withthe hypotonic exposure. Detergents are the most commonlyused cell permeabilization agents and have been used forthe intracellular delivery of biomolecules [25]. Triton X-100has been successfully used to reversibly permeabilize livecells for delivery of optical contrast agents [26]. It wasreported that for the treatment of cells with appropriateconcentrations of Triton X-100, membrane permeabilizationis reversible and membrane integrity typically restoredafter 24 h. The treatment of cells with low concentrationsof digitonin was shown to selectively permeabilize theplasma membrane, leaving the nuclear envelope intact [27].Pore forming bacterial toxins like streptolysin O, whichtemporarily permeabilizes the plasma membrane, have alsobeen investigated for delivery of QDs [28]. However, theactivity of streptolysis O is not stable and it is difficult tocontrol. Saponin, a plant derived glycoside has also been usedfor the permeabilization of cells and for the introduction ofpeptides into the cells [29, 30]. It has been reported thatwhen cells were treated with Saponin, there was minimalrelease of the intracellular macromolecules, minimal damageto the internal architecture and the protein synthesis remainedat comparable levels to that of intact cells [31]. Saponinsreact with membranes rich in cholesterol such as the plasmamembrane and the differential permeabilization of cells hasbeen demonstrated [32]. Our approach is based on the use ofSaponin at low concentrations.

Saponin permeabilization however allows bidirectionaltransport (i.e. into and out of the cell) which can compromisecellular processes via loss of vital intracellular molecularcontent. To ensure unidirectional transport into the cell,Saponin was used in conjunction with hypotonic exposureto enhance osmosis driven transport of QDs into the cellwhile minimizing leakage of intracellular contents out ofthe cell. This method was found to be extremely efficientin accomplishing endocytosis-free delivery of QDs into thecellular cytoplasm while maintaining cell viability. Further,the QDs within the cell were vesicle free and well dispersedwithin the cell and can be used for imaging and biomoleculardelivery.

2. Materials and methods

2.1. Materials

Water soluble CdSe/ZnS core/shell QDs (emission ∼530 nm)were purchased from Ocean Nanotech, LLC. These QDsare a water soluble alloy of CdSe/ZnS core/shell with anamphiphilic polymer coating which in our case is a carboxylic

2

Nanotechnology 24 (2013) 205101 K Medepalli et al

Figure 1. Comparison of confocal microscopy images of H9C2 cells with or without internalized QDs obtained at 60× magnification.(A) phase contrast, (B) fluorescence images of cells incubated at room temperature (RT) showing endocytosis-like uptake of QDs, (C) phasecontrast, (D) fluorescence images of cells incubated at 4 ◦C showing minimal uptake of QDs, (E) phase contrast (control), and(F) fluorescence (control).

acid group. The zeta potential of QDs is from −30 to−50 mV. Their organic layers consist of a monolayer ofoleic acid/octadecylamine and a monolayer of carboxylicacid. The thickness of the total organic layers is about 4 nm.The hydrodynamic size of the QDs is about 20–25 nm. Thecarboxyl group was selected as it can be used for simpleconjugation with several biomolecules. Saponin for reversiblepermeabilization experiments was purchased from Alfa Aesarand deionized water (DI) for the hypotonic experimentswas obtained for a Milli-Q filtration system purchased fromMillipore.

2.2. Cell culture

All experiments utilized a rat cardiomyocyte cell line, H9C2(American Type Culture Collection). The H9C2 cells werecultured in Dulbecco’s Modification of Eagle’s Medium

(DMEM) with 4.5 g l−1 glucose, L-glutamine, and sodiumpyruvate (Fischer Scientific), 10% fetal bovine serum (FBS,Hyclone) and 1% antibiotics (penicillin and streptomycin,Mediatech). Cells were cultured on LabTek chambered coverglass (Fischer Scientific) and multi-well plates (BD Falcon)at a density of approximately 1.2 × 104 cells cm−2. TrypsinEDTA (Mediatech) was used to detach the cells from thesurface for flow cytometry (BD FACS Calibur) studies.

2.3. Reversible permeabilization using hypotonic buffer

H9C2 cells were seeded at a density of approximately1.2 × 104 cells cm−2 and after 24 h the media wasremoved and washed twice with 1× phosphate bufferedsaline (PBS) for cell internalization studies. Hypotonicbuffer solutions at three different ratios (1:1, 3:2, 1:3) wereprepared by mixing S buffer (130 mM sucrose, 50 mM

3

Nanotechnology 24 (2013) 205101 K Medepalli et al

Figure 2. Reversible permeabilization using hypotonic buffer. Comparison of bright field, fluorescence of H9C2 cells treated with 1:1hypotonic buffer ratio for uptake of QDs for ((A), (B)) 2 min, ((C), (D)) 5 min and ((E), (F)) 10 min respectively at 4 ◦C. Bright field andfluorescence images are obtained with 60× magnification using a Nikon A1 confocal system.

KCl, 50 mM potassium acetate, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4) with DIwater at the respective ratios. Cells were treated with QDs(∼10 nM) mixed in the three hypotonic solutions at a pH ofaround 7.4 for 2, 5 and 10 min at 4 ◦C on a rotary shaker.After each time point, the cell culture medium was removedfrom the wells via careful aspiration and the cells were washedtwice with the respective hypotonic buffer solutions, oncewith 1× PBS and thoroughly washed with regular media.They were then incubated for 30–45 min at 37 ◦C for recovery.

2.4. Reversible permeabilization using Saponin in hypotonicbuffer

Saponin solution was prepared at three different concentra-tions of 25, 50 and 75 µg ml−1 in a hypotonic buffer (3:2 ratio

of buffer to DI water). Cells were treated with QDs (∼10 nM)mixed with the above hypotonic Saponin solutions for 2, 5 and10 min at 4 ◦C on a rotary shaker. After each time point, thecells were washed twice with hypotonic S buffer (3:2 ratio)solution, once with 1× PBS and thoroughly washed withregular media. They were then incubated for 30–45 min at37 ◦C for recovery. Control experiments were done by treatingcells with and without QDs (10 nM) in 1× PBS for 10 minand without Saponin or hypotonic conditions. To confirm thecell permeabilization and internalization of the QDs, live cellimaging was carried out using confocal microscopy (NikonA1 confocal system).

2.5. Evaluation of cell viability

Following hypotonic only and Saponin in hypotonicbuffer treatments, reversible permeabilization was confirmed

4

Nanotechnology 24 (2013) 205101 K Medepalli et al

Figure 3. Reversible permeabilization using hypotonic buffer. Comparison of bright field, fluorescence of H9C2 cells treated with 3:2hypotonic buffer ratio for uptake of QDs for ((A), (B)) 2 min, ((C), (D)) 5 min and ((E), (F)) 10 min respectively at 4 ◦C. Bright field andfluorescence images are obtained with 60× magnification using a Nikon A1 confocal system.

by evaluating cell viability using the Live/Dead viabil-ity/cytotoxicity kit (Invitrogen). Cells were cultured in amulti-well plate and the viability study was done following theinstructions in the kit. Briefly, 6 µl of Calcein AM and 15 µlof ethidium homodimer were mixed well in 12 ml of 1× PBSfor 15 min. After the recovery of the cells for 30–45 minfrom hypotonic and Saponin treatments, cells were treatedwith 400 µl of the prepared Calcein–ethidium homodimersolution and incubated at 37 ◦C for 30–45 min. Positiveand negative control experiments were carried out where thecells were treated with Saponin without hypotonic conditionsand without Saponin or hypotonic buffer respectively. Thecells were then carefully washed as mentioned previouslyand were imaged under a fluorescent microscope (Nikoneclipse TE 2000-U). Live and dead cells were distinguished

by the presence of intense uniform green fluorescence(excitation/emission (ex/em) ∼495 nm/∼515 nm) and brightred fluorescence (ex/em) ∼495 nm/∼635 nm) respectively.

2.6. Quantitative and qualitative analysis using fluorescencemicroscopy

All images for the hypotonic and Saponin experiments wereobtained from a Nikon A1 confocal microscope and thebrightness and contrast were equally readjusted for all theimages in Adobe Photoshop. All the images for the cellviability assay were obtained from a Nikon eclipse TE 2000-Ufluorescent microscope and were analyzed for the percentageof live and dead cells using Metamorph software.

5

Nanotechnology 24 (2013) 205101 K Medepalli et al

Figure 4. Reversible permeabilization using hypotonic buffer. Comparison of bright field, fluorescence of H9C2 cells treated with 1:3hypotonic buffer ratio for uptake of QDs for ((A), (B)) 2 min, ((C), (D)) 5 min and ((E), (F)) 10 min respectively at 4 ◦C. Bright field andfluorescence images are obtained with 60× magnification using a Nikon A1 confocal system.

3. Results

3.1. Blocking endocytosis at 4 ◦C

Several studies have indicated that mammalian cellsexposed to QDs at room temperature readily internalizeQDs via endocytosis, whereas exposure at 4 ◦C inhibitedendocytosis [33, 14]. Using H9C2 cells, a representativemammalian cell line, we evaluated the response to QDsdelivered at both room temperature and 4 ◦C. Our resultsindicate that H9C2 cells readily internalize the QDs when theywere incubated at room temperature while minimal uptakewas observed when incubated at 4 ◦C (figure 1). This suggestsan energy dependent endocytosis pathway for the uptake ofQDs at room temperature. Moreover, QDs were internalizedas aggregates, as evident from the punctuated fluorescence,

and were not well distributed in the cells (figure 1). Therefore,to ensure endocytosis-free delivery of QDs, all the QDs’internalization experiments were performed at 4 ◦C.

3.2. Reversible permeabilization using hypotonic buffer

Representative mammalian cells (H9C2 cell lines) werecultured for 24 h and then the cells were treated withQDs in different hypotonic buffer ratios (1:1, 3:2, 1:3)for 2, 5 and 10 min. The strong fluorescence of theQDs (emission ∼530 nm) was utilized for confirming theinternalization and distribution in the cells. Since experimentswere performed at 4 ◦C, the internalization of QDs occursdue to an osmotic loading method. Confocal microscopy wasperformed to image the QDs’ internalized cells and to obtain

6

Nanotechnology 24 (2013) 205101 K Medepalli et al

Figure 5. Cell viability studies. Live/dead cell viability assay showing live cells stained with Calcein AM (green: live cells) and dead cellsstained with ethidium homodimer-1 (red: cells with damaged cell membrane). Cells are treated with (A) different hypotonic buffer ratios(1:1, 3:2, 1:3) for different incubation times (2 min, 5 min and 10 min) at 4 ◦C, (B) different hypotonic Saponin concentrations (25 µg ml−1,50 µg ml−1 and 75 µg ml−1) for different incubation times (2 min, 5 min and 10 min) at 4 ◦C respectively.

a qualitative estimate of the distribution within the cells.Confocal microscopy images of cells with QDs were treatedwith 1:1 (figure 2), 3:2 (figure 3) and 1:3 (figure 4) buffer:DIwater ratios for 2 min (A)–(B), 5 min (C)–(D) and 10 min(E)–(F). Results show that the internalized QDs were welldistributed, as can be seen from the fluorescence signal fromthe cellular cytoplasm. Particularly, the 3:2 buffer:DI waterratio where cells were exposed for 5 min (figures 3(C) and(D)) resulted in the most uniform distribution of internalizedQDs in comparison with other ratios. The evaluation ofcell viability using standard live–dead assays was performedfor the different buffer:DI water ratios: 1:1 (figure 2),3:2 (figure 3) and 1:3 (figure 4) for 2 min, 5 min and10 min respectively. Minimal cell death was observed as aconsequence of exposure to the different hypotonic bufferratios (1:1, 3:2, 1:3) for various incubation times (2, 5, 10 min)(figure 5(A)). However, swelling of the cells was observedwhen a higher ratio buffer with DI was used and also forlonger incubation times.

3.3. Reversible permeabilization using Saponin in hypotonicbuffer

To evaluate if the use of Saponin in conjunction with hypo-tonic exposure improved the internalization and distributionof QDs in cells, H9C2 cells were loaded with QDs inhypotonic conditions in the presence of low concentrationsof Saponin. H9C2 cells were cultured for 24 h prior tothe permeabilization studies. The 3:2 buffer:DI water ratiowas chosen as the preferred ratio for all experiments basedon the intracellular distribution of QDs and the meanfluorescence intensity compared to other ratios. Cells weretreated with three different concentrations of Saponin: 25,50 and 75 µg ml−1 and cells were exposed for 2, 5 and10 min. As described previously, confocal microscopy wasperformed to image the QDs internalized cells and obtain aqualitative estimate of distribution within the cells. Confocalmicroscopy was performed to image the cells with QDs whentreated with 25 µg ml−1 (figure 6), 50 µg ml−1 (figure 7)

7

Nanotechnology 24 (2013) 205101 K Medepalli et al

Figure 6. Reversible permeabilization using Saponin. Comparison of bright field, fluorescence of H9C2 cells treated with 25 µg ml−1 ofSaponin for ((A), (B)) 2 min, ((C), (D)) 5 min and ((E), (F)) 10 min respectively. Bright field and fluorescence images are obtained with60× magnification using a Nikon A1 confocal system.

and 75 µg ml−1 (figure 8) in hypotonic buffer conditionsfor 2 min (A)–(B), 5 min (C)–(D) and 10 min (E)–(F). Ourresults confirm that the QDs were well distributed within thecellular cytoplasm for all three conditions and showed a betterdistribution than with hypotonic exposure alone. Particularly,the 50 µg ml−1 concentration where cells were treated for5 min (figures 7(C) and (D)) shows high QD internalizationand uniform distribution of QDs within the cytoplasm incomparison to the other conditions. It was observed fromconfocal imaging that the use of Saponin in combinationwith hypotonic exposure enhanced the distribution of QDs inthe cells, as is clearly evident from the diffused fluorescenceemerging from inside the cells in the evaluation of cellviability using standard live–dead assays performed for thedifferent Saponin concentrations of 25 µg ml−1 (figure 6),

50 µg ml−1 (figure 7) and 75 µg ml−1 (figure 8) for 2 min,5 min and 10 min respectively. Minimal cell death wasobserved as a consequence of treatment with different Saponinconcentrations (25, 50 and 75µg ml−1) for various incubationtimes (2, 5, 10 min) (figure 5(B)). Finally, to quantify theincreased QD delivery efficiency that can be achieved with theuse of Saponin, flow cytometry was used to estimate the meanfluorescence intensity (MFI) values for cells with internalizedQDs using hypotonic buffer alone and with hypotonic bufferand Saponin, and the results were quantified. Results showcells with QDs internalized under a 3:2 buffer:DI waterratio and exposed for 5 min had a MFI value of 220 ± 15(arbitrary units) whereas for the same conditions the additionof 50 µg ml−1 of Saponin resulted in a MFI value of 342± 31(arbitrary units).

8

Nanotechnology 24 (2013) 205101 K Medepalli et al

Figure 7. Reversible permeabilization using Saponin. Comparison of bright field, fluorescence of H9C2 cells treated with 50 µg ml−1 ofSaponin for ((A), (B)) 2 min, ((C), (D)) 5 min and ((E), (F)) 10 min respectively. Bright field and fluorescence images are obtained with60× magnification using a Nikon A1 confocal system.

3.4. Reversible permeabilization using Saponin in theabsence of hypotonic buffer

To evaluate the role of hypotonic buffer conditions foreffective reversible permeabilization, H9C2 cells weretreated with Saponin in the absence of hypotonic buffer.The concentration of Saponin chosen for this study was125 µg ml−1. This concentration was higher than theconcentrations used for the study with hypotonic buffer(25–75 µg ml−1), however, in the absence of hypotonic bufferlower concentrations of Saponin resulted in poor intracellularQD delivery (supplementary figure 1 available at stacks.iop.org/Nano/24/205101/mmedia). 125 µg ml−1 represents thelowest Saponin concentration where there was appreciableuptake of QDs in the cells. Cells alone without Saponin

served as a negative control whereas 1 mg ml−1 Saponinserved as a positive control. Phase contrast and fluorescencemicroscopy images of standard live–dead assays for cellstreated at different concentrations of Saponin, 0 µg ml−1

(figures 9(A), (B)), 125 µg ml−1 (figures 9(C), (D)), and1 mg ml−1 (figures 9(E), (F)) clearly show increased celldeath following treatment with 125 µg ml−1 of Saponin inthe absence of hypotonic buffer, which is comparable to thecell death seen with treatment with 1 mg ml−1 of Saponin.It can be observed from microscopy images that the cellmembranes were irreversibly damaged during the process.Quantitative evaluation of the percentage of live (green)and dead (red) cells confirms increased cell death followingSaponin treatment (figure 9(G)).

9

Nanotechnology 24 (2013) 205101 K Medepalli et al

Figure 8. Reversible permeabilization using Saponin. Comparison of bright field, fluorescence of H9C2 cells treated with 75 µg ml−1 ofSaponin for ((A), (B)) 2 min, ((C), (D)) 5 min and ((E), (F)) 10 min respectively. Bright field and fluorescence images are obtained with60× magnification using a Nikon A1 confocal system.

4. Discussion

Intracellular delivery of nanomaterials like QDs can beused for applications such as sub-cellular imaging andfor biomolecular transport to manipulate cell signalingmechanisms. Despite the availability of several approachesfor the transport of QDs, the biggest problem remains theirdelivery, free of vesicles such that they are highly diffusedin the cytoplasm. The only technique that has successfullyaccomplished highly dispersed vesicle-free delivery ismicroinjection. However, this technique is laborious and timeconsuming and accomplishes delivery one cell at a time.To overcome obstacles with currently available techniqueswe developed a method for the high throughput reversiblepermeabilization of live cells for the delivery of nanomaterials

like QDs. This technique combines hypotonic exposurewith low concentrations of a cell permeabilization agentsuch as Saponin to accomplish reversible permeabilizationof cell membranes for nanoparticle delivery. Performingthis protocol at 4 ◦C inhibits thermal energy dependentmechanisms like endocytosis and ensures that QD uptake isprimarily due to the reversible permeabilization technique.Hypotonic exposure alone is sufficient for reversiblepermeabilization; however, in combination with Saponintreatment a more efficient and highly dispersed delivery ofQDs is ensured. The mean fluorescence intensities are ameasure of the number of QDs inside the cells while thefluorescence images compare the distribution of QDs withinthe cells.

10

Nanotechnology 24 (2013) 205101 K Medepalli et al

Figure 9. Permeabilization using Saponin without hypotonic conditions. Comparison of bright field and fluorescence images of CalceinAM (green: live cells) and ethidium homodimer-1 (red: cells with damaged cell membrane) co-stained H9C2 cells treated with((A), (B)) 0 µg ml−1 Saponin, cells only (negative control), ((C), (D)) 125 µg ml−1 Saponin without hypotonic buffer, ((E), (F)) 1 mg ml−1

Saponin without hypotonic buffer (positive control) respectively and (G) comparison of mean fluorescence intensity of live/dead cellsstained with Calcein AM (green: live cells) and ethidium homodimer-1 (red: cells with damaged cell membrane) for the same conditions.

We determined experimentally that a 5 min exposureto QDs suspended in a 3:2 buffer to DI water ratio with50 µg ml−1 Saponin produced the best dispersion of theinternalized QDs within the cytoplasm. This protocol alsomaintains cell viability as can be verified from the live–deadassay. The viability study was done after 30–45 min, to beconsistent with the amount of time between recovery andimaging the cells with internalized QDs. The cell viabilitystudy was carried out without QDs’ loading as Calcein AM,which is used for labeling live cells, has an emission around517 nm which would interfere with the QDs’ fluorescenceemission (∼530 nm). Moreover, we have observed that thecells loaded with QDs continued to divide and proliferate afterseveral days without loss of viability.

The use of hypotonic conditions accomplishes two majorfunctions. First, it creates an osmotic pressure gradient thatdrives fluid into the cell. During fluid transport, QDs inthe fluid are also transported into the cells. Secondly, itensures unidirectional transport into the cell. This is importantbecause the cell has different biomolecules and metabolitesthat can diffuse out of the cell when openings are created, orvia aquaporins (water channels) that can cause cell death ordysfunction. The problem with the loss of intracellular contentis even more critical when used in conjunction with Saponin,as holes are created on the cell membrane. Therefore,maintaining the osmotic pressure gradient is important toensure cell viability. The osmotic pressure gradient varieswith time. After 5 min the extracellular and intracellular

environments equilibrate, resulting in a reduction in fluidtransport into the cell and diffusion occurs both into and outof the cells. This explains why the 10 min exposure time doesnot significantly enhance the number of internalized QDs,as seen from the mean fluorescence intensities from flowcytometric analysis. To demonstrate the role of the hypotonicenvironment in maintaining cell viability, experiments wereperformed with Saponin in the absence of a hypotonicenvironment. The low concentrations of Saponin used withouthypotonic buffer (25–75 µg ml−1) did not result in asignificant uptake of QDs and therefore the cell viability datawas not included as this does not represent a viable conditionfor QD delivery. Only when the concentration of Saponinwas raised to 125 µg ml−1 were an appreciable number ofQDs internalized by cells. Live–dead assays performed afterexposure for 5 min at a Saponin concentration of 125 µg ml−1

showed that a majority of the cells were dead, which couldpossibly be due to the leakage of vital intracellular contentsout of the cell in the absence of an osmotic pressure gradientacross the membrane.

This technique is highly efficient for the delivery of QDsfree in the cellular cytoplasm and does not compromise theviability of the cells. Though all experiments were performedusing adherent cells, the technique is applicable for cells insuspension. This technique limits delivery to the cytoplasmand does not accomplish nuclear localization of QDs as theosmotic pressure gradient is across the cell membrane andSaponin only targets cholesterol rich membranes. Specifically,

11

Nanotechnology 24 (2013) 205101 K Medepalli et al

our technique is clearly applicable in in vitro studies where thetracking of a sub-cellular protein or the labeling of individualcellular components is intended. The osmotic loading ofQDs is applicable in in vivo studies such as labeling ofblood leukocytes by selective lysis of red blood cells. Thusour technique has wide applications in the delivery of QDsinto cells for applications such as imaging and biomoleculardelivery. Furthermore, this technique can potentially beextended to the delivery of various other nanomaterials andalso biomolecules into live cells.

5. Summary

In conclusion, we developed a new technique that addressescommon problems associated with nanomaterial deliveryinto cells such as confinement in vesicles and delivery asaggregates. This technique combines the use of a hypotonicbuffer in conjunction with low concentrations of a cellpermeabilization agent, Saponin to accomplish reversiblepermeabilization of cells for delivery of QDs. This techniqueis comparable to microinjection techniques that have beenshown to have the highest success in intracellular QDdelivery but at a significantly higher throughput. Moreovercell viability following this protocol is high and cells survivein culture for extended periods of time and undergo celldivision. This technique has great potential for the delivery ofQDs for both imaging and biomolecular delivery applications.

Acknowledgments

This project was funded via NASA grant No. NNX10AJ36G.The authors would also like to acknowledge confocalmicroscopy facilities provided via NSF grant No. 0814194.

References

[1] Wagner V, Dullaart A, Bock A-K and Zweck A 2006 Theemerging nanomedicine landscape Nature Biotechnol.24 1211–7

[2] Bianco A, Kostarelos K and Prato M 2005 Applications ofcarbon nanotubes in drug delivery Curr. Opin. Chem. Biol.9 674–9

[3] Sarojini H, Medepalli K, Terry D A, Alphenaar B W andWang E 2007 Localized delivery of DNA to the cells byviral collagen-loaded silica colloidal crystals Biotechniques43 216–21

[4] Ferrari M 2005 Cancer nanotechnology: opportunities andchallenges Nature Rev. Cancer 5 161–71

[5] Biju V, Itoh T and Ishikawa M 2010 Delivering quantum dotsto cells: bioconjugated quantum dots for targeted andnonspecific extracellular and intracellular imaging Chem.Soc. Rev. 39 3031–56

[6] Nishikawa M and Huang L 2001 Nonviral vectors in the newmillennium: delivery barriers in gene transfer Hum. Gene.Ther. 12 861–70

[7] Niidome T and Huang L 2002 Gene therapy progress andprospects: nonviral vectors Gene Ther. 9 1647–52

[8] Pannier A K and Shea L D 2004 Controlled release systemsfor DNA delivery Mol. Ther. 10 19–26

[9] Bruchez M, Moronne M, Gin P, Weiss S and Alivisatos A P1998 Semiconductor nanocrystals as fluorescent biologicallabels Science 281 2013–6

[10] Yang R S, Chang L W, Wu J P, Tsai M H, Wang H J,Kuo Y C, Yeh T K, Yang C S and Lin P 2007 Persistenttissue kinetics and redistribution of nanoparticles, quantumdot 705, in mice: ICP-MS quantitative assessment Environ.Health Perspect. 115 1339–43

Kirchner C, Liedl T, Kudera S, Pellegrino T,Munoz Javier A, Gaub H E, Stolzle S, Fertig N andParak W J 2004 Cytotoxicity of colloidal CdSe andCdSe/ZnS nanoparticles Nano Lett. 5 331–8

[11] Derfus A M, Chan W C W and Bhatia S N 2003 Probing thecytotoxicity of semiconductor quantum dots Nano Lett.4 11–8

[12] Pradhan N and Peng X 2007 Efficient and color-tunableMn-doped ZnSe nanocrystal emitters: control of opticalperformance via greener synthetic chemistry J. Am. Chem.Soc. 129 3339–47

Pradhan N, Battaglia D M, Liu Y and Peng X 2006 Efficient,stable, small, and water-soluble doped ZnSe nanocrystalemitters as non-cadmium biomedical labels Nano Lett.7 312–7

[13] Chan W C W and Shuming N 1998 Quantum dotbioconjugates for ultrasensitive nonisotopic detectionScience 281 2016–8

[14] Jaiswal J K, Mattoussi H, Mauro J M and Simon S M 2003Long-term multiple color imaging of live cells usingquantum dot bioconjugates Nature Biotechnol. 21 47–51

[15] Derfus A M, Chan W C W and Bhatia S N 2004 Intracellulardelivery of quantum dots for live cell labeling and organelletracking Adv. Mater. 16 961–6

[16] Okada C Y and Rechsteiner M 1982 Introduction ofmacromolecules into cultured mammalian cells by osmoticlysis of pinocytic vesicles Cell 29 33–41

[17] Courty S, Luccardini C, Bellaiche Y, Cappello G andDahan M 2006 Tracking individual kinesin motors in livingcells using single quantum-dot imaging Nano Lett.6 1491–5

[18] Dubertret B, Skourides P, Norris D J, Noireaux V,Brivanlou A H and Libchaber A 2002 In vivo imaging ofquantum dots encapsulated in phospholipid micellesScience 298 1759–62

[19] Mukherjee S, Ghosh R N and Maxfield F R 1997 EndocytosisPhysiol. Rev. 77 759–803

[20] Sethu P 2006 Microfluidic isolation of leukocytes from wholeblood for phenotype and gene expression analysis Anal.Chem. 78 5453–61

[21] Parekkadan B, Sethu P, van Poll D, Yarmush M L andToner M 2007 Osmotic selection of human mesenchymalstem/progenitor cells from umbilical cord blood Tissue Eng.13 2465–73

[22] Parichehreh V, Estrada R, Kumar S, Bhavanam K, Raj V,Raj A and Sethu P 2011 Exploiting osmosis for blood cellsorting Biomed. Microdevices 13 453–62

[23] Lemoine J L, Farley R and Huang L 2005 Mechanism ofefficient transfection of the nasal airway epithelium byhypotonic shock Gene Ther. 12 1275–82

[24] Djuzenova C S, Krasnyanska J, Kiesel M, Stingl L,Zimmermann U, Flentje M and Sukhorukov V L 2009Intracellular delivery of 2-deoxy-D-glucose into tumor cellsby long-term cultivation and through swelling-activated pathways: implications for radiation treatmentMol. Med. Rep. 2 633–40

[25] Hapala I 1997 Breaking the barrier: methods for reversiblepermeabilization of cellular membranes Crit. Rev.Biotechnol. 17 105–22

[26] van de Ven A L, Adler-Storthz K and Richards-Kortum R2009 Delivery of optical contrast agents using Triton-X100,part 1: reversible permeabilization of live cells forintracellular labeling J. Biomed. Opt. 14 021012

12

Nanotechnology 24 (2013) 205101 K Medepalli et al

[27] Miyamoto K, Yamashita T, Tsukiyama T, Kitamura N,Minami N, Yamada M and Imai H 2008 Reversiblemembrane permeabilization of mammalian cells treatedwith digitonin and its use for inducing nuclearreprogramming by Xenopus egg extracts Cloning StemCells 10 535–42

[28] Nie S 2010 Imaging dynamic cellular events with quantumdots: the bright future Biochem. (Lond.) 32 12

[29] Johnson J A, Gray M O, Karliner J S, Chen C-H andMochly-Rosen D 1996 An improved permeabilizationprotocol for the introduction of peptides into cardiacmyocytes: application to protein Kinase C research Circ.Res. 79 1086–99

[30] Negrutskii B S, Stapulionis R and Deutscher M P 1994Supramolecular organization of the mammalian translationsystem Proc. Natl Acad. Sci. 91 964–8

[31] Hudder A, Nathanson L and Deutscher M P 2003 Organizationof mammalian cytoplasm Mol. Cell. Biol. 23 9318–26

[32] Wassler M, Jonasson I, Persson R and Fries E 1987Differential permeabilization of membranes by Saponintreatment of isolated rat hepatocytes. Release of secretoryproteins Biochem. J. 247 407–15

[33] Kam N W S, O’Connell M, Wisdom J A and Dai H 2005Carbon nanotubes as multifunctional biological transportersand near-infrared agents for selective cancer cell destructionProc. Natl Acad. Sci. USA 102 11600–5

13