Xiangyang Shi et al- Molecular heterogeneity analysis of poly(amidoamine) dendrimer-based mono- and...

8/3/2019 Xiangyang Shi et al- Molecular heterogeneity analysis of poly(amidoamine) dendrimer-based mono- and multifuncti

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Molecular heterogeneity analysis of poly(amidoamine) dendrimer-basedmono- and multifunctional nanodevices by capillary electrophoresis{

Xiangyang Shi, Istvan J. Majoros, Anil K. Patri, Xiangdong Bi, Mohammad T. Islam, Ankur Desai,

T. Rose Ganser and James R. Baker, Jr.*

Received 7th November 2005, Accepted 21st December 2005First published as an Advance Article on the web 18th January 2006

DOI: 10.1039/b515624f

Poly(amidoamine) (PAMAM) dendrimer-based nanodevices are of recent interest in targeted

cancer therapy. Characterization of mono- and multifunctional PAMAM-based nanodevices

remains a great challenge because of their molecular complexity. In this work, various mono- and

multifunctional nanodevices based on PAMAM G5 (generation 5) dendrimer were characterized

by UV-Vis spectrometry, 1H NMR, size exclusion chromatography (SEC), and capillary

electrophoresis (CE). CE was extensively utilized to measure the molecular heterogeneity of these

PAMAM-based nanodevices. G5FA (FA denotes folic acid) conjugates (synthesized from

amine-terminated G5.NH2 dendrimer, approach 1) with acetamide and amine termini exhibit

bimodal or multi-modal distributions. In contrast, G5FA and bifunctional G5FAMTX (MTX

denotes methotrexate) conjugates with hydroxyl termini display a single modal distribution.

Multifunctional G5.AcnFIFA, G5.AcnFAOHMTX, and G5.AcnFIFAOHMTX (Ac

denotes acetamide; FI denotes fluorescein) nanodevices (synthesized from partially acetylated G5

dendrimer, approach 2) exhibit a monodisperse distribution. It indicates that the molecular

distribution of PAMAM conjugates largely depends on the homogeneity of starting materials, the

synthetic approaches, and the final functionalization steps. Hydroxylation functionalization of

dendrimers masks the dispersity of the final PAMAM nanodevices in both synthetic approaches.

The applied CE analysis of mono- and multifunctional PAMAM-based nanodevices provides a

powerful tool to evaluate the molecular heterogeneity of complex dendrimer conjugate

nanodevices for targeted cancer therapeutics.

IntroductionPoly(amidoamine) (PAMAM) dendrimers are a novel class

of advanced synthetic nanoparticles, possessing a core unit,

branches, and multiple terminal groups.1,2 They are synthe-

sized through a stepwise repetitive reaction sequence starting

at the core molecules and giving rise to increasing generations,

with increasing molecular weights and sizes. The interior

cavities of PAMAMs are often used to encapsulate hydro-

phobic or hydrophilic drugs.3,4 In order to achieve better

control of the release rate, imaging, and targeting properties

of drugs, the terminal groups of PAMAMs have been

functionalized with various moieties, such as drugs, biospecific

ligands, and fluorescent tags.512 These dendrimer-based

nanodevices hold great promise for various biomedical

applications, especially for targeted cancer therapy. For

instance, Baker and coworkers6,8 have developed a novel

PAMAM dendrimer-based cancer therapeutic agent carrying

biospecific targeting moieties (e.g. folic acid, denoted as

FA), fluorescent dyes, as well as drugs. These dendrimer

nanodevices are demonstrated to be able to effectivelytransverse vascular pores and directly infuse tumor cells.

Wiener et al. have also developed dendrimerFA conjugates

to target tumor cells expressing high-affinity FA receptors,

and demonstrated that FA-conjugated magnetic resonance

imaging contrast agents represent a promising new approach

for tumor targeting and imaging.13,14 Although the research on

medical applications of dendrimer-based nanodevices con-

tinues progressively, the characterization of these multifunc-

tional dendrimer nanodevices still remains a great challenge.

The comprehensive understanding and analytical characteriza-

tion of the structural heterogeneity of these novel synthetic

nanodevices have not been widely investigated.

PAMAM dendrimers exhibit much narrower poly-dispersity than general synthetic polymers. The polydispersity

of PAMAM dendrimers can be summarized as generational

dispersity (i.e. trailing generations and dimers), skeletal

dispersity (i.e. missing arms and intramolecular loops), and

substitutional dispersity (differences between molecules due to

the non-uniform surface functionalization).15 The former two

dispersities are usually generated by the divergent synthetic

technology,16,17 while the substitutional dispersity is always

present in surface-functionalized PAMAM derivatives.15 For

multifunctional dendrimer-based nanodevices, substitutional

dispersity is often expected due to the multi-step surface

modifications with different functional moieties.

Michigan Nanotechnology Institute for Medicine and BiologicalSciences, University of Michigan, Ann Arbor, MI 48109, USA.E-mail: [email protected]{ Electronic supplementary information (ESI) available: A deconvo-luted normalized electropherogram of G5.NH2 FA conjugates andUV-Vis spectrum analysis of the CE peaks in the electropherogram.See DOI: 10.1039/b515624f

PAPER www.rsc.org/analyst | The Analyst

374 | Analyst, 2006, 131, 374381 This journal is The Royal Society of Chemistry 2006


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Various analytical techniques including, but not limited to,

mass spectrometry (i.e. MALDI-TOF),18 NMR techniques,19

polyacrylamide gel electrophoresis,2024 UV-Vis and fluores-

cence spectroscopy,2528 high performance liquid chromato-

graphy (HPLC),2931 size exclusion chromatography

(SEC),15,32,33 and capillary electrophoresis (CE),15,23,24,3337

have been used to analyze PAMAM dendrimers and deriva-

tives. As opposed to other techniques, CE allows simultaneousevaluation of both purity and charge distribution of the

analyzed materials. Previous studies15,23,24,33 have shown that

CE can be effectively used to estimate molecular distribution

of various functionalized PAMAM dendrimer derivatives.

In this present study, we have synthesized a variety of

generation 5 (G5) PAMAM dendrimer-based functional

nanodevices using two synthetic approaches (See Scheme 1).

Approach 1: synthesis from amine-terminated G5.NH2dendrimers; the synthesized materials include monofunctional

G5FA conjugates (terminated with amine, acetamide, and

hydroxyl groups), and bifunctional G5.FAOHMTX (MTX

denotes methotrexate) conjugates terminated with hydroxyl

groups. Approach 2: synthesis from partially acetylated G5dendrimers; the synthesized materials include a set of mono-

and multifunctional G5.Acn FA, G5.Acn FIFA, G5.Acn

FAOHMTX, and G5.Acn FIFAOHMTX nanodevices

(FI denotes fluorescein). SEC was used to evaluate the

molecular weights of these dendrimer nanodevices, while

UV-Vis spectrometry was employed to detect the functional

moieties upon dendrimer surface modification. NMR techni-

ques were also utilized to confirm the dendrimer surface

functionalization and estimate the average number of surface

functional moieties. CE was extensively used to evaluate the

molecular distribution of these dendrimer-based nanodevices,

since the charge distribution and electrophoretic mobility often

change upon dendrimer surface conjugation. The applied CE

analysis provides a unique way to evaluate the molecular

distribution and heterogeneity of the dendrimer-based nano-

devices for targeted cancer therapy. To our best knowledge,this is the first extensive research report regarding the novel

application of CE for the analysis of complex dendrimer-based

mono- and multifunctional nanodevices.

Experimental

Materials

Ethylenediamine (EDA)-cored PAMAM G5.NH2 dendrimer

was synthesized in the Michigan Nanotechnology Institute for

Medicine and Biological Sciences, University of Michigan

(Ann Arbor, MI) and was completely characterized by various

techniques.9,38 The number of terminal amine groups was

determined to be 110. The molecular weight and polydispersitydata are listed in Table 1. Acetic anhydride, glycidol, MTX,

absolute methanol, triethylamine, dimethyl sulfoxide (DMSO),

FA, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC),

and all the other chemicals and solvents were obtained from

Aldrich (Milwaukee, WI) and used as received. Phosphate

buffer (pH = 2.5, 50 mM) and water used for CE analysis were

obtained from Agilent Technologies (Waldbronn, Germany)

and used as received. Water used in all the other experiments

Scheme 1 Schematic representation of the synthetic approaches used to synthesize mono- and multifunctional dendrimer nanodevices.

This journal is The Royal Society of Chemistry 2006 Analyst, 2006, 131, 374381 | 375


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was purified by a Milli-Q Plus 185 water purification

system (Millipore, Bedford, MA) with resistivity higher

than 18 MV cm. Regenerated cellulose dialysis membranes

(MWCO = 10,000) were acquired from Fisher (Fairlawn, NJ).

Synthesis of G5FA conjugates (terminated with amine,acetamide, and hydroxyl groups) and G5.FAOHMTX

conjugates terminated with hydroxyl groups using approach 1

The amine-terminated G5.NH2 dendrimer was conjugated

with FA using our published procedures (conjugate 1).6 Based

on the 1H NMR integration values and the comparison

between the aromatic signals of FA and aliphatic region of

dendrimer methylene protons, we estimated that an average of

5.0 FA moiety molecules were present per dendrimer. The

remaining amines on the dendrimer surface were modified

with acetic anhydride to give a complete dendrimer acetamide

functionalized with the FA moiety (conjugate 2). The 1H

NMR of this acetylated dendrimer shows merging methyleneprotons and an additional singlet at 1.8 ppm for the terminal

COCH3 groups.32 Alternatively, the amine terminated

G5FA conjugate was further hydroxylated with glycidol

(conjugate 3), followed by esterification with methotrexate

using EDC coupling chemistry (conjugate 4).9 All physico-

chemical parameters of the above dendrimer conjugates are

shown in Table 1.

Synthesis of G5.AcnFA, G5.Ac

nFIFA, G5.Ac

nFAOH

MTX, and G5.AcnFIFAOHMTX conjugates using

approach 2

This set of G5 dendrimer conjugates were synthesized from

partially acetylated G5 dendrimers. A list of the synthesized

conjugates (conjugate 5, 6, 7, 8) is shown in Table 1.

Details can be found in a previous report.9 The numbers of

the conjugated FA, FI, and MTX were determined using 1H

NMR, UV-Vis spectrometry, and SEC (See Table 1).

Characterization techniques

SEC analysis. SEC was used to determine the absolute

molecular weights of the as-prepared PAMAM dendrimer

conjugates. SEC experiments were performed using an Alliance

Waters 2690 separation module (Waters Corporation,

Milford, MA) equipped with a Waters 2487 UV absorbance

detector (Waters Corp.), a Wyatt Dawn DSP laser photometer

(Wyatt Technology Corporation, Santa Barbara, CA), and an

Optilab DSP interferometric refractometer (Wyatt Technology

Corporation). Citric acid buffer (0.1 M) with 0.025% sodium

azide in water was used as a mobile phase. The pH of the

mobile phase was adjusted to 2.74 using NaOH, and the flowrate was maintained at 1 mL min21. Sample concentration was

kept at approximately 2 mg mL21 and 100 mL of analytes was

injected for all measurements. Molar mass moments of the

PAMAM dendrimers were determined using Astra software

(version 4.7) (Wyatt Technology Corp.).

UV-Vis spectrometry. UV-Vis spectra of dendrimer con-

jugates were collected using a Perkin Elmer Lambda 20 UV-

Vis spectrometer. All the dendrimer conjugates were dissolved

in pH 2.5 phosphate buffer (50 mM) and their pHs were

adjusted to 2.5 using 0.1 M phosphoric acid. The final con-

centrations of the dendrimer conjugates were 1 mg mL21.

NMR measurements. 1H NMR spectra of PAMAM

dendrimer conjugates were recorded on a Bruker DRX 500

nuclear magnetic resonance spectrometer. Dendrimer samples

were dissolved in D2O at a concentration of 5 mg mL21 before

NMR measurements.

CE analysis. An Agilent Technologies (Waldbronn,

Germany) CE instrument was used in this work. Unmodified

quartz capillaries were purchased from Polymicro

Technologies (Phoenix, AZ). Voltage was kept at 20 kV for

all the separations. On-capillary UV diode-array detection was

used, operating at four different wavelengths depending on the

functional moieties conjugated on dendrimer surfaces. Sampleswere introduced by hydrodynamic injection at a pressure of

50 mbar. Silanized capillaries24 (id 100 mm) with total length of

78.5 cm and effective length of 70 cm were employed. Some

of the experiments were performed using a short capillary

(id 100 mm) with total length of 48.5 cm and effective length of

40 cm. The capillary temperature was maintained at 40 uC.

Before use, the silanized capillary was initialized by rinsing

with 0.2 M H3PO4 for 15 min, deionized water for 15 min, and

the running buffer (50 mM, pH 2.5 phosphate buffer) for

another 15 min. Prior to each injection, the capillary was

rinsed with a sequence of 0.2 M H3PO4 (3 min), deionized

water (3 min), and the running buffer (10 min). G5 PAMAM

Table 1 Physicochemical parameters of dendrimer and dendrimer conjugates

Dendrimer Conjugate Mn Mw/Mn Number of functional moieties

G5.NH2 26 330 1.032 N/AG5.NH2FA 1 28 090 1.084 3.8(FA)

a, 5.0(FA)b

G5.FAAc 2 32 870 1.172 3.8(FA)a, 5.0(FA)b

G5.FAOH 3 40 210 1.108 3.8(FA)a, 5.0(FA)b

G5.FAOHMTX 4 44 270 1.120 3.8(FA)a,5.0(FA)b, 9.3(MTX)a,10.0 (MTX)b

G5.Ac75 29 900 1.021 N/AG5.Ac75FA 5 31 400 1.027 5.5 (FA)b, 5.5 (FA)c

G5.Ac75FIFA 6 34 100 1.003 4.7 (FI)c, 5.5 (FA)b, 5.5 (FA)c

G5.Ac82FAOHMTX 7 36 730 1.006 5.7 (FA)a, 4.5 (FA)b, 4.8 (FA)c, 5.5 (MTX)a

G5Ac82FIFAOHMTX 8 39 550 1.008 5.8 (FI)a, 5.7 (FA)a,4.5 (FA)b, 4.8 (FA)c, 5.5 (MTX)a

a Based on the differences between the measured Mn by SEC, which is divided by the molecular mass of the incoming substituent. b Based onthe differences between the integrals of 1H NMR signals associated with dendrimers and the surface moieties. c Based on UV-Vis spectroscopyanalysis using the concentration calibration curve of free FA.



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conjugates were dissolved in the running buffer and the sample

solutions were adjusted to pH 2.5 using 0.1 M phosphoric

acid to give a final concentration of 1 mg mL21. All dendrimer

conjugate samples contained 0.05 mg mL21

2,3-diaminopyr-

idine (2,3-DAP) as an internal standard to normalize the

migration time of analytes.24 Each sample was run 5 times to

confirm the reproducibility. The CE electropherograms were

deconvoluted using PEAKFIT program (SYSTAT SoftwareInc., Point Richmond, CA 94804) to perform compositional

analysis of the dendrimer conjugate species.

Results

Size exclusion chromatography

SEC allows for the detection of the absolute molecular weights

of the as-synthesized dendrimer conjugates. The polydisper-

sities of all dendrimer conjugates are very close to 1.000 (See

Table 1), indicating the narrowly dispersed nature of PAMAM

dendrimers. Based on the molar mass difference between G5

dendrimer conjugates and the starting materials, one can

calculate the number of functionalized groups or moieties. Forinstance, the number of MTX moiety molecules attached on

conjugate 4 was calculated to be 9.3 based on its molar mass

difference from conjugate 3, which is in agreement with that

calculated based on NMR integration. The molar masses

of G5FA conjugates are in the order of G5.FAOH

(conjugate 3) > G5.FAAc (conjugate 2) > G5.NH2FA

(conjugate 1). We also found that an incomplete hydroxylation

reaction took place when comparing the molar mass difference

between 1 and 3. Based on the calculation, the number of

terminal glycidol moiety of3 was 164, which was less than 210

according to the ideal stoichiometry. Namely, there are 46

secondary amines remaining intact under the experimental

conditions. This observation is consistent with our previous

results showing that incomplete hydroxylation always appears

regardless of the number of dendrimer generations.15,33 The

incomplete hydroxylation may contribute to the structural

heterogeneity of the dendrimer conjugates (vide infra). The

same SEC measurements were carried out for a set of mono-

and multifunctional G5 dendrimer nanodevices synthesized

using approach 2 (conjugates 5, 6, 7, 8, see Table 1). From

SEC results, G5 dendrimer nanodevices synthesized using both

approach 1 and approach 2 exhibited similar polydispersity,

mainly due to the limitation of SEC technique that functions

based on the sizes of the macromolecules.

UV-Vis spectrometry

UV-Vis spectrometry was used to detect the functional

moieties attached on dendrimer molecules. Fig. 1 shows the

UV-Vis spectra of G5FA conjugates terminated with amine

(1), acetamide (2), and hydroxyl (3) groups and a bifunctional

G5.FAOHMTX conjugate (4). It is clear that curves 1, 2,

and 3 exhibit the specific maximum absorption peak at

287 nm, which is ascribed to the contribution of the FA

moiety. This suggests the successful conjugation of FA with

dendrimers (UV-Vis spectrum of free FA not shown). The

UV-Vis absorption profiles of the G5FA conjugates are

almost similar regardless of the type of the terminal groups.

For the bifunctional G5.FAOHMTX nanodevice, due to the

structural similarity of FA and MTX,39 their absorptions

overlap. Therefore, it is difficult to differentiate the maximumabsorption of MTX and FA moieties. The maximum absorp-

tion at 303 nm may result from the contribution of both FA

and MTX moieties. The shoulder present at 243 nm is virtually

related to the contribution of the MTX moiety as compared

with the UV-Vis spectrum of free MTX (not shown). The

UV-Vis spectra of dendrimer conjugates demonstrates the

successful conjugation of the functional moieties. The absorp-

tion features also direct the optimal selection of the on-

capillary UV detection wavelengths for CE measurements

in order to achieve maximum sensitivity. The same UV-Vis

spectroscopy studies were performed on the mono- and

multifunctional G5 dendrimer nanodevices synthesized using

approach 2. Details can be found in the previous literature.9

Overall, by using UV-Vis spectrometry, one can confirm the

successful conjugation of imaging, targeting, and therapeutic

agents onto the G5 dendrimer surfaces.

Capillary electrophoresis

Analysis of G5FA conjugates synthesized using approach 1.

For the analysis of G5FA conjugates, four different UV

wavelengths (200 nm, 210 nm, 250 nm, and 280 nm) were

selected based on the UV-Vis spectroscopic studies. The

selection of the 280 nm wavelength is expected to increase

the detection sensitivity of the FA moiety. Fig. 2 shows the

normalized electropherograms of G5FA conjugates termi-nated with amino (1), acetamide (2), and hydroxyl (3) groups.

It is apparent that conjugate 3 exhibits a relatively homo-

geneous migration peak, while 1 and 2 display at least bimodal

distribution. Estimation of the FA moiety distribution in

dendrimer conjugates can be accomplished by performing off-

line UV-Vis spectrum analysis of individual migration peaks

using Agilent software. Inset of Fig. 3a shows the UV-Vis

spectrum assignments of the corresponding migration peaks

shown in Fig. 3a and b. A dendrimer material associated

with peak 2 displays much higher absorption at 280 nm

(characteristic maximum absorption of the FA moiety) than

that corresponding to peak 1, indicating that peak 2-related

Fig. 1 UV-Vis spectra of the G5FA conjugates with different

terminal groups synthesized using approach 1. Curves 1, 2, 3, and 4

represent the spectra of G5.NH2FA (1), G5.FAAc (2), G5.FAOH

(3), and G5.FAOHMTX (4), respectively.



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material possesses more FA moiety molecules than that related

to peak 1. This phenomenon can also be easily differentiatedby comparing the absorbances of peak 1 and peak 2 at

different detection wavelengths. At 210 nm, peak 1-related

dendrimer conjugate displays higher absorbance than that

related to peak 2, while at 280 nm this is reversed. The

deconvoluted electropherograms of G5.FAAc (2) at 210 nm

(Fig. 3a) and 280 nm (Fig. 3b) are also shown, which were used

for quantitative analysis of peak compositions (Table 2). The

same analysis was applied to G5.NH2 FA conjugate (1). It

appears that peak 1-related dendrimer conjugate possesses

more FA moiety molecules than peak 2-associated dendrimer

material (See supporting information{).

Analysis of G5FAMTX conjugate (4) synthesized using

approach 1. G5.FAOHMTX bifunctional nanodevice (4)

was also analyzed using CE. To determine different moieties

attached onto dendrimer surfaces, the suitable wavelengths

(210 nm, 240 nm, 300 nm, 350 nm) of on-capillary UV-Vis

diode array detector were selected. The selection was based on

the UV-Vis spectrum of 4 collected in the CE running buffer

(pH 2.5 phosphate buffer, 50 mM) (Fig. 1). 210 nm was used to

detect the dendrimer aliphatic backbone, while 240 nm and

300 nm were used for MTX and FA moiety analysis, respec-

tively. Fig. 4 shows a typical CE electropherogram of 4. One

can clearly observe that conjugate 4 exhibits a monomodal

symmetrical peak. The material was confirmed to be pure, as

no impurity peaks were present in the electropherogram even

at a long migration time up to 50 min. To determine the real

composition of the conjugate, an off-line UV-Vis spectrometry

analysis was performed using the Agilent software (Fig. 4,

inset). The UV-Vis spectrum of peak 2-related conjugate

species (Fig. 4, inset) is consistent with the UV-Vis spectrum of

conjugate 4 measured by UV-Vis spectrometer (See Fig. 1),

further indicating that peak 2 corresponds to the studied

bifunctional G5 conjugate.

Fig. 2 Normalized electropherograms of G5FA conjugates termi-

nated with amino (1, curve 1), acetamide (2, curve 2), and hydroxyl (3,

curve 3) groups detected at 210 nm. The first peak is related to the

internal standard 2,3-DAP, which exhibit an average migration time of

12.14 min (7 runs). The CE was run using a silanized silica capillary (id

100 mm) with effective length of 70 cm and total length of 78.5 cm.

Fig. 3 Deconvoluted normalized electropherograms of G5.FAAc conjugate (2) at 210 nm (a) and 280 nm (b). The off-line UV-Vis spectra of

individual migration peaks are shown in the inset of (a).

Table 2 Compositional estimation of the studied dendrimer conjugates based on the deconvolution of CE electropherograms at210 nm using PEAKFIT program

Dendrimer G5.NH2FA (1) G5.FAAc (2)

Peak # 1 2 3 1 2 3Migration time/min 17.12 17.88 20.51 19.05 22.60 24.50Peak area 48.2 20.6 17.6 61.2 18.0 10.0Percentage (%) 55.8 23.8 20.4 68.6 20.2 11.2

Fig. 4 A typical electropherogram of G5.FAOHMTX conjugate

(4) detected at 210 nm using a silanized silica capillary (id 100 mm) with

effective length of 40 cm and total length of 48.5 cm. Peaks 1 and 2

correspond to the internal standard 2,3-DAP and conjugate 4,

respectively. An off-line UV-Vis spectrum of peak 2 is also shown in

the inset.



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Analysis of mono- and multifunctional G5 conjugates

synthesized using approach 2. In another experiment, a set of

mono- and multifunctional G5 nanodevices were analyzed

using CE. These materials were synthesized from partially

acetylated G5 dendrimer. Fig. 5 shows the CE electrophero-

grams of G5.Ac75, G5.Ac75 FA (5), G5.Ac75 FIFA (6),

G5.Ac82FAOHMTX (7), and G5Ac82FIFAOHMTX

(8) conjugates. It is clear that all dendrimer conjugates display

a single peak, indicating their relatively good monodispersity.

Conjugate 6 shows a longer migration time than both G5.Ac75and conjugate 5, which is probably due to its lower charge/

mass ratio and structural complexity after conjugation with

the FI moiety. G5.Ac82FAOHMTX (7) and G5Ac82FIFAOHMTX (8) conjugates were synthesized from a

partially acetylated G5.Ac82 dendrimer. After conjugation

with FA and/or FI, the intermediate products were hydro-

xylated by reacting with glycidol, followed by conjugation

with MTX to give the final MTX-conjugated nanodevices.

Both conjugates (7 and 8) show narrower peaks than those

of G5.Ac75, 5, and 6 (please note that the comparison is

reasonable because the difference between G5.Ac75 and

G5.Ac82 is small enough and can be neglected). The narrow

peaks of7 and 8 might result from the hydroxylation reaction,

which has been shown to mask the polydispersity of

dendrimers in our previous work.15,33 In a sense of molecular

heterogeneity, hydroxylation actually improves the mono-

dispersity of the multifunctional dendrimer nanodevices.

The CE system holds a diode array UV detector and an off-

line UV-Vis spectral analysis feature, which allows identifyingthe individual CE peaks. By comparing the UV-Vis spectra of

analyzed species with those recorded using conventional UV-

Vis spectrometer, one can identify the composition of each CE

peak related species. Fig. 6a shows the CE electropherograms

of a tri-functional PAMAM nanodevice (8) detected at 210,

300, and 495 nm. The material displays a single peak at all

wavelengths indicating that FI, FA, and MTX moieties are

homogeneously present. A magnified electropherogram

(Fig. 6b) shows the distribution of the FI moiety. Off-line

UV spectra of peak 1 and peak 2 (Fig. 6a, inset) further

confirmed that the peak compositions are related to the

internal standard 2, 3-DAP and the analyzed nanodevice (UV

spectrum of the tri-functional nanodevice peak is consistentwith the spectrum measured using UV-Vis spectrometer).9 All

the mono- and multifunctional G5 dendrimer nanodevices

were analyzed in the same way. They all displayed a

monodisperse molecular distribution.

Discussion

Molecular distribution of dendrimer-based nanodevices

regarding polymer polydispersity does not increase signifi-

cantly after surface multi-step conjugation, as verified by SEC

measurements (Table 1). This is because SEC is only based on

polymer size distribution that does not change significantly

upon dendrimer surface conjugation. As opposed to otheranalytical techniques (i.e. SEC, UV-Vis spectrometry, and

NMR) that can only detect the average molecular physico-

chemical parameters, CE provides a unique way to detect the

distribution of molecular species based on the differences in

their electrophoretic mobilities resulting from the change of

their charge/mass ratios. Possible factors affecting the mole-

cular distribution of functional dendrimer conjugates are

briefly discussed as follows.

Fig. 5 CE electropherograms of G5 dendrimer conjugates synthe-

sized using approach 2 detected at 210 nm. The first peak is related

to the internal standard 2,3-DAP. The CE was run using a silanized

silica capillary (id 100 mm) with effective length of 40 cm and total

length of 48.5 cm. Curve 1: G5.Ac75; curve 2: G5.Ac75FA (5); curve 3:

G5.Ac75 FIFA (6); curve 4: G5.Ac82 FAOHMTX (7); curve 5:

G5Ac82FIFAOHMTX (8).

Fig. 6 CE electropherograms of a tri-functional PAMAM nanodevice (G5Ac82FIFAOHMTX, 8) detected at 210, 300, and 495 nm (a). A

magnified electropherogram detected at 495 nm is shown in (b). Off-line UV-Vis spectra of peak 1 (internal standard) and peak 2 (nanodevice) are

shown in the inset of (a). The CE was run using a silanized silica capillary (id 100 mm) with effective length of 40 cm and total length of 48.5 cm.



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(1) The classification of dendrimer terminal groups influ-

ences the surface moiety distribution of dendrimer conjugates.

We previously demonstrated that the dispersities of dendri-

mers could be magnified by acetylation and masked by

hydroxylation for amine-terminated PAMAM dendrimers.15

For all materials synthesized using approach 1 (starting from

amine-terminated G5.NH2), more prominent FA moiety dis-

tribution can be observed in G5.FAAc (2) than that ofG5.NH2FA (1) (Fig. 2), while G5.FAOH (3) and G5.FA

OHMTX (4) (Fig. 2 and 4) only show a mono-modal

distribution. In the case of hydroxyl-terminated dendrimer

conjugates, because of the incomplete hydroxylation and the

existence of the resulting secondary terminal amines, the very

close number of terminal hydroxyl groups masks the molecular

differences between individual dendrimer conjugate molecules.

This is expected to result in a mono-modal distribution of

dendrimer conjugate species. These results are consistent with

our previous report.15 Similarly, multifunctional G5.Ac82FA

OHMTX (7) and G5Ac82FIFAOHMTX (8) nanodevices

synthesized using approach 2 display narrower CE peaks than

those of G5.Ac75, 5, and 6 that do not possess hydroxyl terminalgroups (Fig. 5). This further suggests the dispersity masking

effect of hydroxylation reaction for dendrimer conjugation.

Please note that peak 2-related dendrimer conjugate in the

electropherogram of G5.FAAc (2) display a higher concentra-

tion of FA moiety molecules than peak 1-related material, which

is opposed to peak composition comparison of G5.NH2FA (1)

conjugate (Fig. 3 and supporting information{). This may be

reasoned as follows: in the separation of 2, the hydrophobic

interaction between the silanized capillary surface (methyl

groups) and 2 may be stronger than the non-specific electrostatic

interaction, which makes conjugate 2 containing more hydro-

phobic FA moiety molecules migrate slower than those contain-

ing less FA moiety molecules.(2) The synthetic approaches. Two synthetic approaches

were employed to synthesize mono- and multifunctional G5

dendrimer nanodevices. Compared with approach 1 which the

conjugation reaction started from amine-terminated G5.NH2dendrimer, in approach 2 partially acetylated G5 dendrimer

was instead used as a starting material. The high local

concentration of amine groups of G5.NH2 makes it highly

reactive, thereby significantly distributing the conjugated

moiety molecules. Whereas, after partial acetylation reaction,

the small portion of amine groups remaining on the G5

dendrimer surface can preserve the homodispersity of the

dendrimer for subsequent bioconjugation.9 Consequently,

mono- and multifunctional dendrimer nanodevices synthesizedusing approach 2 always display a monomodal distribution,

which is opposed to those dendrimer conjugates synthesized

from an amine-terminated G5 dendrimer (approach 1). Please

note that the partial acetylation reaction is crucial for one to

produce targeted monodisperse dendrimer conjugates. The

acetylation reaction is very fast. One has to add dilute acetic

anhydride solution slowly to a dendrimer solution under

vigorous stirring in order to achieve highly monodispersed

partially acetylated G5 dendrimer. If the preparation

procedure is not appropriate, highly dispersed G5.Acn can be

obtained. This definitely influences the polydispersity of the

final G5 PAMAM nanodevices.

Conclusions

In summary, various mono- and multifunctional dendrimer

nanodevices were synthesized either from amine-terminated

or from partially acetylated G5 dendrimers and were charac-

terized using SEC, UV-Vis spectrometry, 1H NMR, and CE.

CE was extensively used to evaluate the molecular distribution

of the as-synthesized dendrimer conjugates. The CE results

show that the molecular distribution of the as-synthesized

dendrimer nanodevices largely depends on the classification of

the final functionalization step and the synthetic approaches.

For dendrimer conjugates synthesized from amine-terminated

G5 dendrimers, hydroxyl-terminated monofunctional and

multifunctional nanodevices display a monomodal distribu-

tion, while amino and acetamide-terminated G5FA con-

jugates show multimodal distributions. Mono- and

multifunctional G5 dendrimer conjugates synthesized from

partial acetylated G5 dendrimers always display a monomodal

distribution. It indicates that partial acetylation is critical to

preserve the homodispersity of dendrimers for subsequent

bioconjugation reactions. The applied CE analysis of mono-

and multifunctional PAMAM-based nanodevices provides an

alternative approach to evaluate the molecular heterogeneity

of complex dendrimer conjugate nanodevices, which are being

used for targeted cancer therapy.

Acknowledgements

We thank Chunyan Chen for her valuable remarks and

suggestions. This work is financially supported by the National

Cancer Institute (NCI), National Institute of Health (NIH),

under the contract # NOI-CO-97111.

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