Ajit SharmaDillip K. MohantyAnkur DesaiRiaz Ali

Department of Chemistry,Central Michigan University,Mt. Pleasant, MI, USA

A simple polyacrylamide gel electrophoresisprocedure for separation of polyamidoaminedendrimers

A simple, inexpensive, and rapid electrophoresis technique was developed for use as aroutine tool for evaluating purity of polyamidoamine (PAMAM) dendrimers. A variety offactors influencing migration of generations 0–7 dendrimers on nongradient polyacryl-amide gels were evaluated. The low generation dendrimers were found to be very sen-sitive to diffusion during or after electrophoresis. The proposed method incorporatessteps that minimize diffusion, in order to obtain improved resolution and sensitivity,especially for the lower-molecular-weight dendrimers. This was accomplished byinclusion of a dendrimer fixation step with glutaraldehyde and performing the electro-phoresis separation, fixation, staining, and destaining at 4�C. PAMAM dendrimerseparation was studied under basic and acidic conditions. Electrophoresis underacidic conditions gave increased resolution and sensitivity over separation at alkalinepH. Oligomers and trailing generations could be clearly separated and visualized underthese conditions. The smallest PAMAM dendrimer, generation 0, was visible at 1.5 �gunder the optimized acidic conditions. With slight modifications, this technique shouldbe applicable to separation of other water-soluble dendrimers.

Keywords: Nongradient polyacrylamide gel / Polyamidoamine dendrimerDOI 10.1002/elps.200305540

1 Introduction

Dendrimers are synthetic polymer-based nanoparticles,which are becoming increasingly important in biochem-ical research. Due to their high density of surface func-tional groups, many synthetic and natural moleculeshave been attached either covalently or noncovalently todendrimers, producing conjugated products that haveapplications in gene and drug delivery and as diagnosticreagents [1]. The most popular class of dendrimers usedin biological research is the polyamidoamine (PAMAM)dendrimers. These water-soluble macromolecules arecommercially available and are synthesized by adding arepeat unit to the growing polymer. The smallest PAMAMdendrimer is referred to as generation 0 (G0) and increas-ingly larger ones are called G1, G2, G3 and so on. PAMAMdendrimers have many features common with proteins.They have well-defined composition, molecular weights,and topology. The backbone of their chains are made upof amide linkages similar to the peptide bonds of proteins.

Like proteins, PAMAM dendrimers have been reportedto undergo some folding, shape and size changes inresponse to changes in their environment, such as alteredsolvent type and pH.

Dendrimer separation and characterization is important intheir synthesis as well as in studies on their interactionswith other molecules. A number of techniques have beenused, including small angle neutron and X-ray scattering,electron and atomic force microscopy, spectroscopy andelectrophoresis. Electrophoresis is a preferred method inbiochemical research. Electrophoresis equipment andexpertise is widely available. In addition, simple modifica-tions of analytical conditions allow one to separate anycharged, water-soluble dendrimers of varied shapes anddimensions (e.g., nucleic acid and polylysine dendrimers).The end signal is visual and result interpretation is rela-tively easy. Electrophoresis is also an inexpensive tech-nique that does not require sophisticated instrumentationand highly skilled operators. For these reasons, it hasbecome an increasingly important tool for separatingPAMAM dendrimers of various generations as well asPAMAM complexes and conjugates [2–4]. However, thereis limited information on the separation and detection ofPAMAM dendrimers by PAGE [2]. In the present investiga-tion, the influence of various electrophoresis parameterson the separation of full generation ethylenediamine (EDA)core PAMAM dendrimers by PAGE was evaluated. The

Correspondence: Dr. A. Sharma or Dr. D. K. Mohanty, Depart-ment of Chemistry, Central Michigan University, Mt. Pleasant,MI 48859, USAE-mail: sharmla@cmich.edu or mohanldk@cmich.eduFax: +989-774-3883

Abbreviations: EDA, ethylenediamine; PAMAM, polyamido-amine; TBE, Tris-borate-EDTA

Electrophoresis 2003, 24, 2733–2739 2733

2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0173-0835/03/1608–2733 $17.50�.50/0

Gen

eral

2734 A. Sharma et al. Electrophoresis 2003, 24, 2733–2739

main objective was to develop a simple, inexpensiveseparation technique that could be used routinely forassessing dendrimer purity or for quality control duringsynthesis of PAMAM dendrimers.

2 Materials and methods

2.1 Materials

PAMAM dendrimers were either purchased from Aldrich(Milwaukee, WI, USA) or were a generous gift from Den-dritic Nanotechnologies Limited (Mt. Pleasant, Ml, USA).All other chemicals were from Sigma Chemical (St. Louis,MO, USA).

2.2 PAGE of PAMAM dendrimers

Native gel electrophoresis on 10% or 15% acrylamidegels was performed either under basic or acidic condi-tions. Minigels (10 cm�8 cm�0.75 mm) were usedthroughout this study. Electrophoresis was performed ona Dual Mini-Vertical Slab Gel Electrophoresis Unit, whichallows gel cooling by running water (Sigma Chemical).Samples were applied on the anode side and migratedtowards the cathode. Gels were photographed with aKodak DC 40 digital camera and images analyzed withKodak Digital Science 1D image analysis software (East-man Kodak, Rochester, NY, USA). Electrophoresis on gra-dient gels was carried out on 4–20% precast polyacryl-amide minigels made in Tris-HCl, pH 8.5 (BioExpress,Kaysville, UT, USA). These gels were not compatible withthe Sigma electrophoresis system and were therefore runon a Mini-PROTEAN 3 electrophoresis system (Bio-RadLaboratories, Hercules, CA, USA).

2.2.1 Basic conditions

A 5% stacking gel was made in 0.063 M Tris-HCl, pH 6.8.The final concentrations of ammonium persulfate andTEMED were 0.06% and 0.063%, respectively. For gen-erations G0–G4 a 15% resolving gel was used, while forthe higher generations G5–G8 a 10% gel was used. Theresolving gel was made in TBE buffer (90 mMTris, pH 8.3,80 mM boric acid, and 2.5 mM EDTA). The final con-centrations of ammonium persulfate and TEMED were0.1% and 0.038%, respectively. Loading buffer was 40%sucrose and 2% methylene blue. Samples were mixedwith equal volumes of loading buffer for application ontothe gel. Electrophoresis buffer was TBE buffer (90 mM Tris,pH 8.3, 80 mM boric acid, and 2.5 mM EDTA) to which wasadded 0.19 M glycine. Electrophoresis was performed at4�C, typically at 200 V, for 1–2 h. After separation, the gel

was placed in 0.25 M bicarbonate buffer for about 5 min atroom temperature. It was then transferred to a fresh solu-tion of bicarbonate buffer (containing 0.5 M glutaralde-hyde) and incubated at 4�C for 1 h. After fixation, the gelwas rinsed with deionized water and placed in Coomas-sie blue stain (0.2% dye made in 50% methanol/10%acetic acid) in the cold (4�C). Although most of the bandsshowed up in approximately 4 h, staining was typicallyperformed overnight in order to visualize faint bands.Destaining was carried out in 10% methanol/10% aceticacid solution in the cold. Usually 3–4 changes after everyhour in the cold destaining solution yielded a clear back-ground.

2.2.2 Acidic conditions

The 5% stacking gel was made in glacial acetic acid/KOHsolution (final concentrations 0.75% and 120 mM, respec-tively; pH 5.9). Ammonium persulfate and TEMED con-centrations were 0.7% and 0.06%, respectively. In addi-tion, sodium bisulfite (0.008%) was added to enhance po-lymerization at low pH. Resolving gel was made in glacialacetic acid/KOH solution (final concentrations 13.25%and 30 mM, respectively; pH 2.9). The concentrations ofpersulfate and bisulfite were similar to that of stackinggel. TEMED concentration was increased to 0.6%. Underthese conditions, polymerization took about 30–60 min.Electrode buffer was 0.16% acetic acid containing 0.6%�-alanine. The rest of the procedure was similar to thatunder alkaline conditions.

3 Results and discussion

The synthesis scheme for an EDA core G0 is shownbelow.

Reaction of EDA with methyl acrylate occurs viaMichael’saddition to form a polyester-terminated intermediate thatis referred to as the “half generation”. The half generationdendrimer is then reacted with excess EDA to produce afull generation G0. This process is repeated to form highergenerations. Determination of the purity of low generationdendrimers is therefore of utmost importance in order toavoid carry over to higher generations.

Electrophoresis 2003, 24, 2733–2739 Polyamidoamine PAGE 2735

3.1 Electrophoresis under alkaline conditions

Brothers et al. [2] first described the use of polyacryl-amide gel electrophoresis for PAMAM separation. Fullgeneration EDA core PAMAM dendrimers were separatedin a 5–40% gel using a TBE buffer (90 mM Tris, pH 8.3,80 mM boric acid, and 2.5 mM EDTA). Minigels were runat constant voltage (200 V), typically for 90 min. Reversepolarity was used for analysis of the positively charged fullgeneration PAMAM dendrimers. Samples were loaded insucrose with methylene blue used as the tracking dye.Gels were stained overnight with Coomassie BrilliantBlue R-250 (made in 40% methanol and 7% acetic acid)and destaining was performed in 5% methanol-7% aceticacid.

Figure 1. (A) Electrophoresis of PAMAM dendrimerson 15% native PAGE under nonoptimized alkaline con-ditions. Lane 1, generations G0–G4 mixture (ladder);2, generation G1; 3, generation G4; 4, generation G3;5, generation G2; 6, generation G5; 7, generation G0.Electrophoresis was performed at 200 V for 60 min. Inall figures, the cathode is at the bottom of the figure.(B) Electrophoresis of PAMAM dendrimers on 10%native PAGE under nonoptimized alkaline conditions.Lane 1, generations G5-G8 mixture (ladder); 2, genera-tion G8; 3, generation G7; 4, generation G6; 5, genera-tion G5. Electrophoresis was performed at 200 V for75 min. (C) Electrophoresis of PAMAM dendrimers on15% native PAGE under optimized alkaline conditions.Lane 1, generations G0–G4 mixture (ladder); 2, genera-tion G4; 3, generation G3; 4, generation G2; 5, genera-tion G1; 6, generation G0. Electrophoresis was per-formed at 200 V for 60 min. Electrophoresis of PAMAMdendrimers on 10% native PAGE under optimized alka-line conditions. Lane 1, generation G5; 2, generation G6;3, generation G7; 4, generation G8; 5, generations G5–G8 mixture (ladder). Electrophoresis was performed at200 V for 90 min.

Since gradient gels are relatively difficult to prepare in areproducible manner and are commercially expensive,only nongradient polyacrylamide gels were used in thisinvestigation. Preliminary experiments showed that a15% acrylamide concentration was a suitable range forresolving dendrimer generations from G0 (Mr 517) to G4(Mr 14 215) while a 5–10% gel was useful for resolv-ing G5 (Mr 28 826) to G8 (Mr 233 383). A 5% stacking gelwas used throughout this study. Stock PAMAM solutionsof generations 0–4 in methanol were diluted with TBE buf-fer and applied to a 15% nondenaturing resolving gel.This gel was polymerized in TBE buffer, pH 8.3 and wasoverlaid with a 5% stacking gel (made in Tris-chloride,pH 6.8). Electrophoresis conditions were similar tothose employed by Brothers et al. [2]. Each lane con-tained 10 �g dendrimer. Electrophoresis was performedat constant voltage (200 V, 60 min) and at room tempera-ture. The gel was stained overnight with Coomassie blue(made in 50% methanol-10% acetic acid mixture). Thiswas followed by overnight destaining with 10/10 metha-nol-acetic acid (without shaking) in order to obtain a clearbackground. A typical electropherogram of PAMAM den-drimers from generation 0 to 5 is shown in Fig. 1A. Lane 1shows a mixture of dendrimers G0–G4, each at 10 �g,while lanes 2–7 demonstrate separation of individual gen-erations, also at 10 �g. Sharpness of the bands increasedwith increase in molecular weight of the dendrimer. TheG0 band was rarely visible. When it did appear asshown in Fig. 1A, it showed up as a highly diffused halo-like structure (“ladder”, lane 1 and lane 7). Generation G1was also often undetectable and appeared as a halo(lanes 1 and 2). Generation G2 was always seen but ittoo sometimes appeared as a halo (lanes 1 and 5). It isinteresting to note that dendrimers in a mixture (ladder)may show different electrophoresis characteristics fromindividually applied samples. For example, in Fig. 1A,generations G1 and G2 have a halo-appearance in theladder and not when individually applied (compare lanes1 with 2 and 5). G3 and higher generations, however,always appeared as clear bands (Fig. 1A). In addition, ingels stained for only a few minutes, the bands in theladder appeared more intense than those in other lanes,although the amount of each generation was the samein all lanes. The reason for this is unclear. Separation ofhigher generations on a 10% resolving gel, under similarconditions is shown in Fig. 1B. G5–7 were clearly resolvedand showed sharp, distinct bands. G8 and higher genera-tions remained in the 5% stacking gel. As before, bandswere sharper with increasing size of the dendrimer.

A variety of electrophoresis conditions were evaluated inorder to obtain improved separation, especially for thelower generation dendrimers. The molecular weights ofG0–G4 are less than 15 000 and they have a flat, floppy

2736 A. Sharma et al. Electrophoresis 2003, 24, 2733–2739

conformation compared to robust spherical shapes of thehigher generations [5]. The size of EDA-core PAMAM den-drimers are similar to proteins of comparable molecularweights. For example, insulin (Mr 5800) and G3 (Mr 6909)are about 3 nm in dimension, cytochrome c (Mr 13 000)and G4 (Mr 14 215) are about 4 nm while hemoglobin(Mr 64 500) and G5 (Mr 28 826) are about 5.4 nm. There-fore, procedures used in protein electrophoresis and de-tection were evaluated on dendrimer separation.

To detect any potential interactions between dendrimerand tracking dye (methylene blue), separations were per-formed with and without dye in the sample buffer. Nodifferences were found (results not shown). The problemswith separation of the low generation dendrimers (G0–G2)are most likely due to diffusion, which may occur during orafter electrophoresis. Conditions that minimize diffusionwere studied. When electrophoresis was performed inthe cold (4�C), slight improvements in band sharpnesswas obtained. However, migration rates were less thanat room temperature and G0 was still not visible. G1 oftenappeared as a very faint band. The effects of electricalvariables on dendrimer electrophoresis were evaluated.Keeping all other conditions similar to those used inFig. 1A, electrophoresis was carried out under variouscurrents and voltages. Running the gel at high voltagesor currents (greater than 200 V or 20 mA) led to morediffuse bands. Under the above conditions, a constantcurrent of around 15 mA or constant voltage of about150–200 V gave good separations within a reasonableamount of time (1–2 h). Under these conditions, whenelectrophoresis was performed at constant current, volt-age increased with time while at constant voltage, currentdecreased with time. A combination of low temperatureand low voltage (or current) gave gels where generationG0 was clearly seen, even at 5 �g, and without any halo(results not shown). These results indicate that the lowgeneration PAMAM dendrimers, especially G0 and G1,were very sensitive to diffusion during electrophoresis.Since Joule heating is a major cause of sample diffusion,any conditions that reduce heat formation during electro-phoresis will reduce diffusion. These include using lowercurrents or voltages and running the gel in the cold.

Another potential cause of sample diffusion is the post-electrophoretic steps of staining and destaining. In pro-tein electrophoresis, the sample is precipitated afterseparation, thereby converting it into an insoluble form.This prevents diffusion of the proteins, thus keeping theprotein bands sharp and resolved during the stainingstep. A low pH and alcohol combination (e.g., aceticacid/methanol) is often used in protein fixation. Strongerfixatives commonly employed include trichloroaceticacid, sulfosalicylic acid and aldehydes (formaldehyde

and glutaraldehyde). In the present method, the stain(Coomassie blue) was made in 50% methanol/10%acetic acid mixture, and staining was typically carriedout overnight. Destaining also involved using mixtureswith methanol, typically lasting for a few hours. SincePAMAM dendrimers are readily soluble in methanol,sample diffusion may be significant during the stainingand destaining steps. In order to reduce post-electro-phoresis diffusion, simultaneous fixing and staining wasperformed by making the Coomassie blue stain in morenonpolar solvent mixtures, in which water was eithercompletely eliminated or methanol was replaced byhigher alcohols like butanol. However, none of thesecombinations gave significant improvements in bandsharpness or intensity. Therefore, inclusion of a fixingstep prior to staining was evaluated, Unlike proteins,PAMAM dendrimers do not denature and aggregate.Therefore, dendrimer fixation was carried out usingcross-linking reagents. Aldehyde fixatives, such as glu-taraldehyde, react with surface amines and create cova-lent cross-links between protein molecules resulting inprecipitate formation. Since the pKa of the terminal pri-mary amines of PAMAM dendrimers is about 9–10, apH of 9.5 (bicarbonate buffer) was used for dendrimercoupling. At this pH, a significant portion of the terminalamines would be unprotonated and act as reactivenucleophiles towards the dialdehyde. PAMAM dendri-mers possess anywhere from 4 (G0) to 2048 (G9) surfaceamine groups. Dendrimer samples G0, G2 and mixture(G0–G4) were run at 15 mA constant current and roomtemperature. After electrophoresis, one set of gels werefixed and stained at room temperature and another setwas fixed and stained in the cold (4�C). Gels were fixedwith either 20 mM or 500 mM glutaraldehyde (pH 9.5)for 1 h, followed by overnight staining. The control gelthat was not fixed showed G2 and higher generations,clearly. G1 in the mixture appeared as a halo while G0was not visible. Fixing with low concentrations of glutar-aldehyde (� 20 mM) at room temperature, showed shar-per bands without any halos and G0 was faintly visible.Higher glutaraldehyde concentration (�500 mM), undersimilar conditions, did not show any significant improve-ments in band sharpness, but G0 was clearly visible.When fixing and staining were performed at 4�C, thebands were much sharper and darker compared to fix-ing/staining at room temperature. Gels fixed with 0.5 M

glutaraldehyde at 4�C, followed by staining in the cold,gave the best resolution. No halos were seen and G0was dark and sharp. The mixture of dendrimer genera-tions 0–4 were well resolved into five sharp and distinctbands (results not shown). These results suggest thatthe post-electrophoresis steps for dendrimer detectioncontribute significantly to sample diffusion and poor res-olution.

Electrophoresis 2003, 24, 2733–2739 Polyamidoamine PAGE 2737

Gels were next run under the optimized conditions de-scribed above. These conditions included electrophore-sis separation at low temperatures and at low voltage(or current), followed by a one-hour fixing in cold 0.5 M

glutaraldehyde (pH 9.5) and subsequent overnight stain-ing at 4�C. Destaining was performed by immersing thegel in 10%/10% methanol-acetic acid mixture at 4�C.Destaining time was kept to a minimum, wherein suffi-cient contrast was obtained between bands and back-ground (typically, 3–4 h). As described above, all of theseconditions were found favorable towards reducing sam-ple diffusion during or after electrophoresis. Under theseconditions sample stacking was probably due to sodiumions (in sodium EDTA) and Tris cations, the former actingas the leading ion with the latter as the trailing ion. Addi-tion of 0.19 M glycine to the TBE buffer led to furtherimprovements in dendrimer separation, probably dueto improved stacking with glycine or the trailing cation. Atypical separation of G0–G4 obtained under these opti-mized conditions is shown in Fig. 1C. There were signifi-cant improvements over the nonoptimized conditions(Fig. 1A). All the bands obtained were sharp. The laddershowed five distinct bands (G0–G4). Lower generationdendrimers, G0–G2, were always visible and did not showany halos. As before, band sharpness was proportional tomolecular weight of the dendrimer. Changing the ionicstrength of the Tris-borate buffer or eliminating borate(and/or EDTA) from the buffer did not show any improve-ments in dendrimer separation. Separation of G5–G8 on a10% resolving gel under these optimized conditions isshown in Fig. 1D. G5–G7 bands were sharper comparedto separation under nonoptimized conditions (Fig. 1B).

3.2 Electrophoresis under acidic conditions

Citrate buffer (0.1 M, pH 3.0) was previously used forseparation of EDA-core PAMAM dendrimers on a 5–40%polyacrylamide gel and at constant voltage of 200 V [2].When we diluted dendrimer samples with citrate buffer,the sample aggregated and formed precipitates. Theextent of precipitation increased with increasing size ofthe dendrimer. Sample dilution with glycine buffer (0.1 M,pH 3.0) did not cause any precipitation, but the electro-phoresis yielded grossly distorted bands, especially withthe lower generations. Panyim and Chalkley [6] used 0.9 M

acetic acid buffer and tube gels made in acetic acidfor separation of histones at constant current. Electro-phoresis of PAMAM dendrimers under these conditionsdid not give any significant improvements over alkalineconditions (results not shown). In addition, the increasedgel polymerization time as well as the volatility of aceticacid (in the gel) led to problems in preparing wells in slabgels.

A modified procedure recommended by Reisfeld el al. [7]was developed to include a stacking gel and to overcomethe problems mentioned above. The stacking gel wasmade in glacial acetic acid/KOH solution. Ammoniumpersulfate was increased from 0.1% to 0.7% and TEMEDwas increased from 0.04% to 0.6%. In addition, sodiumbisulfite was added to enhance polymerization at low pH[8]. Resolving gels were made in glacial acetic acid/KOHsolution. The concentrations of persulfate, TEMED andbisulfite were similar to that of stacking gel. Electrode buf-fer was 0.16% acetic acid containing 0.6% �-alanine.Electrophoresis was carried out under the optimized con-ditions described above. Under these conditions, theleading ion was K� and the trailing ion was �-alanine. Atypical gel obtained under optimized acid pH is shown inFig. 2A. For comparison, the same dendrimer sampleswere also run under the optimized alkaline conditions

Figure 2. (A) Electrophoresis of PAMAM dendrimers on15% native PAGE under optimized acidic conditions.Lane 1, generations G0–G4 mixture (ladder); 2, generationG0; 3, generation G1; 4, generation G2; 5, generation G3;6, generation G4. Electrophoresis was performed at 200 Vfor 60 min. (B) Comparison with alkaline conditions. Thesame samples in (A) were run under optimized basicconditions. Lane 1, generations G0–G4 mixture (ladder);2, generation G0; 3, generation G1; 4, generation G2;5, generation G3; 6, generation G4. Electrophoresis wasperformed at 2000 V for 60 min. (C) Electrophoresis ofPAMAM dendrimers on 10% native PAGE under opti-mized acidic conditions. Lane 1, generations G5–G8 mix-ture (ladder); 2, generation G8; 3, generation G7; 4, gen-eration G6; 5, generation G5. Electrophoresis was per-formed at 150 V for 90 min.

2738 A. Sharma et al. Electrophoresis 2003, 24, 2733–2739

used in Fig. 1C (Fig. 2B). Sample size for the gels shown inFig. 2 was reduced from 10 �g to 5 �g. As shown in Fig. 2,the bands obtained under acidic conditions were oftensharper than in alkaline conditions. All generations wereclearly visible even at 5 �g sample size. No halos wereobserved. More importantly, acidic conditions gave betterresolution than separation at basic pH. As is evident fromFig. 2A, under acidic conditions, G2–G4 (lanes 4–6)showed additional bands. Some of these bands have ahigher mobility while others travel slower than the mainband. For example, G4 showed four bands, two of whichtraveled faster and one that was slower than the main G4band. The additional bands represent higher order oligo-mers and trailing generations, which are formed duringsynthesis. Larger aggregates are due to double amidationof EDA between two dendrimers. Trailing generations areformed as a result of incomplete removal of EDA after theamidation step. The residual EDA can then act as a newinitiator core and form a trailing generation. In the gelshown in Fig. 2A, the slower moving band (G4D, lane 6)is likely a dimer of G4, with a mobility similar to G5 whilethe two faster moving bands (G3TG and G2TG) representits trailing generations that migrate like a G3 or a G2,respectively [2]. Similarly, under acidic conditions, thetrailing generations of G3 and G2 are also seen in lanes 4and 5, respectively. These species are not well resolvedunder alkaline conditions (Fig. 2B). Electrophoretic sepa-ration of G5–G7 under optimized acidic pH is shown inFig. 2C. A trailing generation of G5 (G4TG) is clearly visi-ble (lane 5). Similarly, trailing generations of G6 and G7are also seen in lanes 4 and 5. The dimer of G6 (G6D)can also be observed (lane 4), The proposed acidic elec-trophoresis procedure thus provides a simple, inexpen-sive technique for semiquantitative assessment of den-drimer purity. For synthesis and purification of dendri-mers, it is essential that these contaminants be clearlyresolved, especially for the lowest generations.

Figure 3 shows separation of various amounts of a den-drimer sample made by spiking G1 with G0 and G2. Lane1 was a mixture of G0–G4, while lanes 2,3, and 4 con-tained 1.25 �g, 0.75 �g, and 0.25 �g of G2, 5 �g, 3 �g,and 1.0 �g of G1 and 2.5 �g, 1.5 �g, and 0.5 �g of G0,respectively. It is evident that separation under acidicconditions (Fig. 3A) is also more sensitive than electro-phoresis under basic conditions (Fig. 3B). Although it isnot clear from the gel image shown in Fig. 3, G0 wasseen in the acidic gel even at 1.5 �g while under alkalineconditions, the sensitivity of G0 was 2.5 �g.

Another major advantage of running PAMAM dendrimersunder acidic conditions is demonstrated in Fig. 4. Figures4A and B show separation of PAMAM-OH under acidicand basic conditions, respectively. Lane 1 is the ladder.

Figure 3. Electrophoresis of spiked G1 under optimized(A) acidic and (B) basic conditions. Lane 1, generationsG0–G4 mixture, each at 5 �g (ladder); 2, mixture of G0(2.5 �g), G1 (5 �g) and G2 (1.25 �g); 3, mixture of G0(1.5 �g), G1 (3 �g) and G2 (0.75 �g); 4, mixture of G0(0.5 �g), G1 (1 �g) and G2 (0.25 �g). Electrophoresiswas performed at 200 V for 60 min.

Figure 4. Electrophoresis of G4 PAMAM-OH under opti-mized (A) acidic and (B) basic conditions. Lane 1, genera-tions G0–G4 mixture, each at 5 �g (ladder); 2, G4 (5 �g);3,G5 (5 �g); 4, G4–OH (5 �g); 5,G4–OH (10 �g); 6, G4–OH(15 �g); 7, G5 (5 �g). Electrophoresis was performed at200 V for 80 min.

Under acidic conditions, dendrimers with terminal func-tional groups that are uncharged, such as those contain-ing hydroxyl groups (PAMAM-OH), can be well-separated(Fig. 4A). This is not possible under basic conditions(Fig. 4B). Lanes 4, 5, and 6 show 5. 10 and 15 �g, respec-tively, of generation 4 PAMAM-OH. The molecular weightof G4 PAMAM-OH (Mr 14 279) is almost equivalent to G4PAMAM (with amine termini, Mr 14 215). Under acidicconditions, G4 PAMAM-OH travels slower than G4PAMAM (compare lane 2 with lane 5, Fig. 4A) and showsa mobility similar to G5 PAMAM (lane 3) which has a mo-lecular weight of 28 826. The reduced mobility of G4PAMAM-OH compared to G4 PAMAM is expected, dueto its decreased positive charge density. Consequently,longer times are required for separation of PAMAM-OHdendrimers (gels shown in Fig. 4 were run for 80 mininstead of 60 min). However, under acidic conditions, thecore secondary and tertiary amines of G4-OH are pro-

Electrophoresis 2003, 24, 2733–2739 Polyamidoamine PAGE 2739

Figure 5. (A) Electrophoresis of spiked dendrimer sam-ples on a 4–20% gradient gel, under nonoptimized alka-line conditions and fixed with glutaraldehyde. Lane 1,generations G0-G6 mixture, each at 5 �g (ladder); 2, mix-ture of G0 and G1 (each at 5 �g); 3, mixture of G1 andG2 (each at 5 �g); 4, mixture of G2 and G3 (each at 5�g). Electrophoresis was performed at 200 V for 60 min.(B) Electrophoresis of spiked dendrimer samples on a4–20% gradient gel, under nonoptimized alkaline con-ditions, without glutaraldehyde fixing. Lane 1, mixture ofG0 and G1 (each at 5 �g); 2, mixture of G1 and G2 (eachat 5 �g); 3, mixture of G2 and G3 (each at 5 �g); 4, gen-erations G0-G6 mixture, each at 5 �g (ladder). Electro-phoresis was performed at 200 V for 60 min. Sampleswere not fixed.

tonated and provide sufficient charge density for its elec-trophoretic mobility. On the other hand, under basic con-ditions these groups are mostly unprotonated, resulting inlower mobility of G4-OH (lanes 4–6, Fig. 4B). The ability ofacidic gels to resolve oligomers and trailing generationseven for PAMAM-OH dendrimers is also clearly demon-strated in Fig. 4A. These contaminants can be seen evenat 5 �g G4 PAMAM-OH (lane 4). It should be noted thatthe G4-OH, because it lacks terminal amine groups, wasprobably not fixed by glutaraldehyde.

It should be noted that the diffusion-related artifacts dis-cussed above are also observed on gradient gels. Pre-vious reports did not demonstrate effective separation oflower generations PAMAM dendrimers (G0 and G1) evenwith 5–40% gels [2]. Separation of spiked dendrimersamples on a 4–20% gradient gel, under conditionsrecommended by Brothers et al., is shown in Fig. 5. In

the absence of a fixation step, broad, halo-like bandswere obtained for G0, G1 and even G2 (Fig. 5B). Glutaral-dehyde fixation led to improved band resolution (Fig. 5A).

4 Concluding remarks

The most critical factor for PAMAM dendrimer electro-phoresis was sample diffusion that led to poor resolution,especially for the smaller lower generations. Steps thatminimized dendrimer diffusion either during or after elec-trophoresis improved resolution and sensitivity. The useof low temperature (4�C) for separation and post-electro-phoresis manipulations led to improved dendrimer sepa-ration. Fixation was another important step that was notperformed in earlier reports on dendrimer PAGE. The bisaldehyde glutaraldehyde was an effective fixative forpolyamidoamine dendrimers. PAMAM dendrimer electro-phoresis may be performed under either basic or acidicconditions. Acidic conditions had several advantages in-cluding improved resolution and sensitivity, and the abilityto resolve and detect PAMAM dendrimers that do nothave surface charged groups, such as PAMAM-OH.

The project described was supported by Grant-No. 1R15GM065975-01 from National Institute of Health. Its con-tents are solely the responsibility of the authors and donot necessarily represent the official views of NIH. Theauthors also greatly acknowledge the financial support(FRCE and PRIF grants) from Central Michigan University.

Received March 31, 2003

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