Parag Kolhe et al- Preparation, cellular transport, and activity of polyamidoamine-based dendritic...

8/3/2019 Parag Kolhe et al- Preparation, cellular transport, and activity of polyamidoamine-based dendritic nanodevices with

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Biomaterials 27 (2006) 660669

Preparation, cellular transport, and activity of

polyamidoamine-based dendritic nanodevices with a

high drug payload

Parag Kolhea, Jayant Khandarea,b, Omathanu Pillaib, Sujatha Kannanb,Mary Lieh-Laib, Rangaramanujam M. Kannana,

aDepartment of Chemical Engineering and Material Science, and Biomedical Engineering, Wayne State University, 5050,

Anthony Wayne Drive, Detroit, MI-48202, USAbDepartment of Pediatrics (Critical Care Medicine), Childrens Hospital of Michigan, Wayne State University, Detroit, MI-48201, USA

Received 28 February 2005; accepted 20 June 2005Available online 27 July 2005

Abstract

Dendrimers are emerging as a relatively new class of polymeric biomaterials with applications in drug delivery, and imaging.

Achieving a high drug payload in dendrimers, and understanding the therapeutic effect of the dendrimerdrug conjugates are

receiving increasing attention. A high drug payload nanodevice was obtained by covalent conjugation of ibuprofen to a

polyamidoamine (PAMAM-G4-OH) dendrimer. Using DCC as a coupling agent, 58 molecules of ibuprofen were covalently

conjugated to one molecule of generation 4 PAMAM-OH dendrimer. Cellular entry of the fluoroisothiocynate (FITC)-labeled

dendrimerdrug conjugate was evaluated in vitro by using human lung epithelial carcinoma A549 cells by flow cytometry, confocal

microscopy and UV/Visible spectroscopy. The pharmacological activity of the dendrimeribuprofen conjugate was compared to

pure ibuprofen at various time points by measuring the suppression of prostaglandin E2. Significant amounts of the conjugate

entered the cells rapidly within 15 min. Suppression of prostaglandin was noted within 30 min for the dendrimerdrug conjugates

versus 1 h for the free ibuprofen. The results suggest that dendrimers with high drug payload improve the drugs efficacy by

enhanced cellular delivery, and may produce a rapid pharmacological response. These dendrimerdrug conjugates can potentially be

further modified by attaching antibodies and ligands for targeted drug delivery.

r 2005 Elsevier Ltd. All rights reserved.

Keywords: PAMAM dendrimers; Dendrimerdrug conjugates; Drug delivery; Ibuprofen; Cellular transport

1. Introduction

Advances in polymer science have led to intelligent

material design for achieving spatial and temporalcontrol of drug delivery even at a molecular level.

Biological and cellular functions of living organisms are

strictly designed on a hierarchy of size-scales varying

from centimeters to nanometers. At the most funda-

mental level, function and structure necessary for life,

result from specific molecular structures and shapes in

the nanometer scale. Hence, molecular level strategies to

target, deliver, detect, diagnose and treat diseases could

be more therapeutically efficacious compared to asystemic approach [1]. The unique nanoscale architecture

of dendrimer offers an extraordinary interfacial and

functional advantage for drug-delivery applications at

all levels in the biological hierarchy [2].

Dendrimers have generated a great deal of interest for

various applications due to their exceptional structural

properties such as monodispersity ($1.0), high density

of peripheral functional groups, well-defined globular

shape ($20 nm) and multivalency [3,4]. These salient

ARTICLE IN PRESS

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0142-9612/$- see front matter r 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biomaterials.2005.06.007

Corresponding author. Tel.: +1 313577 3879;

fax: +1 313577 3810.

E-mail address: [email protected] (R.M. Kannan).
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features make dendrimers potential alternatives to

traditional polymers in a wide range of applications,

especially as nanodevices for controlled and targeted

delivery of therapeutic compounds. Variety of dendritic

polymers have been synthesized with a hydrophobic

core or a hydrophilic shell for diverse applications. In

the past, various strategies have been devised to modifydendrimers with drug molecules, genetic materials,

targeting agents, dyes and imaging agents, either by

encapsulation or conjugation [414]. By conjugating

appropriate targeting moieties, drugs, and imaging

agents to dendritic polymers, smart drug-delivery

nanodevices can be developed that can target, deliver,

and monitor the progression of therapy. Drugs can be

conjugated to dendritic nanodevices through either ester

or amide linkage, which can be hydrolyzed inside the cell

by endosomal or lysosomal enzymes. Encapsulation of

drugs in PEGylated dendrimers can lead to enhanced

permeation and retention (EPR) of the drug [8]. The

nanoscale branching architecture of the dendrimers

provides them with several advantages over linear

polymers, nanoparticles and liposomes such as rapid

cellular entry, reduced macrophage uptake and target-

ability [15,16].

Although significant strides have been made in the

design of drug-delivery systems, developing a system

that can eventually reach the desired target and deliver

the drug still remains a challenge. Most of the recent

strategies in literature have focused on the use of

dendrimers to target chemotherapeutic agents using

cell-surface receptors [17]. On the other hand, these

versatile vehicles remain unexplored in number oftherapeutic areas where the target site is intracellular,

such as pain management and inflammation. The main

issues associated with the use of dendrimers for drug

delivery include, overcoming cytotoxicity especially in

the case of cationic dendrimers, improving drug pay-

load, and understanding the mechanism and dynamics

of intracellular transport. The objective of the present

study was two-fold (i) achieving a high drug payload,

and (ii) demonstrating the potential of PAMAM G4-

OH dendritic nanomaterials to deliver the drug intra-

cellularly. Studies have shown the potential to target

chemotherapeutic agents to tumor cells mostly with

cationic dendrimers [8,9,18]. However, neutral dendri-

mers such as PAMAM-G4-OH (which are expected to

be less cytotoxic than the NH2 terminated dendrimer)

have not been widely investigated for the drug delivery.

We have recently shown that PAMAM-G4-OH-termi-

nated dendrimer was less cytotoxic than cationic

PAMAM-G4-NH2-terminated dendrimer [19].Previously, we have reported the synthesis and

evaluation of PAMAMNH2 dendrimeribuprofen

complexes involving ionic interaction between amine

groups of dendrimer and carboxyl groups on ibuprofen

[20]. Unlike the drugdendrimer complex, the covalently

linked drugdendrimer conjugates would be more

stable in vivo, thus prolonging drug circulation

and tissue delivery. In this paper, we report the synthe-

sis and evaluation of OH-terminated PAMAM dendri-

meribuprofen conjugates with a high drug payload

for enhanced cellular delivery. Ibuprofen is a non-

steroidal anti-inflammatory drug (NSAID), which

shows side effects such as renal dysfunction and

gastrointestinal hemorrhage when delivered by conven-

tional drug-delivery systems. Improving the efficacy

of ibuprofen by using dendrimerdrug conjugates as

an advanced drug-delivery system to achieve a

high intracellular concentration of the drug at the site

of action can potentially minimize the systemic side

effects.

2. Experimental

2.1. Materials and synthesis

Fluoroisothiocyanate probe FITC was purchased from

Fluka chemical company. PAMAM-G4-OH terminal dendri-

mer (average molecular weight $14,279Da) was purchased

from Sigma-Aldrich. Structural features of dendrimer used in

this study are summarized in Table 1. Ibuprofen-USP

(racemic(7) form) and dicyclohexylcarbodiimide (DCC) were

purchased from Aldrich chemical company. Interleukin (IL-

1b) and lipopolysaccharide were purchased from Sigma, USA.

ELISA kit for prostaglandin estimation was purchased from

Cayman Chemical Company. Dialysis membrane of molecular

weight cut-off of 3500 Da was obtained from Spectrapor.

Solvents dimethyl sulphoxide (DMSO), dimethyl formamide

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Table 1

Molecular properties of PAMAM-G-4-OH-terminated dendrimer and ibuprofen conjugation ratios

Avg. Mw

(g/mol)

No. of end

groups

b % Mole of

ibuprofen in

conjugate

b % Wt. of

ibuprofen in

conjugate

bAverage No. of

ibuprofen in

conjugate

bAverage Mw with

ibuprofen in the

conjugates (g/mol)

PAMAM-G4-OH 14,279a 64 98 47.44 58 25,183

Ibuprofen 206 1

aReported by Tomalia et al. (1990).bEstimated by 1H NMR integration method.

P. Kolhe et al. / Biomaterials 27 (2006) 660669 661


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(DMF) were purchased from Fischer Scientific. All other

chemicals used were of analytical grade.

2.2. Synthesis of dendrimer ibuprofen conjugate

PAMAM-G4-OH terminal dendrimer (0.75 g, 0.05 mM)

and ibuprofen (1.5 g, 7.28 mM) were dissolved in anhydrous

DMSO. (The molar ratio of the drug to the dendrimer wascalculated on the basis of molecular weight and number of end

groups of the dendrimer). To this solution, DCC (1.5g,

7.28 mM) was added as a coupling agent and the reaction was

stirred continuously for 3 days at room temperature. The re-

action mixture was filtered to remove dicyclohexylurea (DCU)

formed during the reaction. The solution was further dialyzed

(dialysis membrane of molecular weight cut-off 3500 Da)

against DMSO for 24 h to remove free ibuprofen and DCC.

Excess solvent was removed under vacuum at room tempera-

ture to obtain dendrimeribuprofen conjugates. Repurification

was carried out using diethyl ether to remove any unreacted

ibuprofen. The number of ibuprofen molecules conjugated per

mole of PAMAM-G4-OH was estimated using

1

H NMR.

2.3. Synthesis of PAMAM-G4-OH ibuprofen FITC

conjugates

FITC (0.02 g, 0.051 mM) was first conjugated with glutaric

acid (0.067g, 0.051 mM) to form a carboxyl-terminated FITC

using DCC (0.09 mg) as a coupling agent. Further, PAMAM-

G4ibuprofen conjugates (0.5 g, 0.0019 mM) and FITCglu-

taric acid moiety (0.078g, 0.014 mM) were dissolved in

anhydrous DMSO and DCC (0.04 g, 0.0019 mM) was added

to it. DCC acts as a coupling agent to couple FITCglutaric

acid with unreacted hydroxyl groups of PAMAM-G4 in

PAMAM-G4ibuprofen conjugates, to form an ester bond.

The reaction mixture was stirred for 5 days at roomtemperature and filtered to remove N, N0-dicyclohexylurea.

The solution was filtered and dialyzed against DMSO for 24 h

to remove unreacted FITCglutaric acid and DCC. The

contents inside the dialysis membrane were removed and

further purified with acetone to remove free FITCglutaric

acid. Absence of free FITC in the conjugate was verified by

TLC using chloroform and methanol (ratio 1:1) as mobile

phase. The product was dried under vacuum to obtain

PAMAM-G4ibuprofenFITC conjugates.

2.4. Gel permeation chromatography (GPC)

GPC analysis was carried out on Waters GPC instrumentequipped with manual injector and UV detector interfaced to

Breeze software. The mobile phase used was 0.05 M NaHCO3/

0.1M NaOH/deionized water (50:23:27) with a pH of 11.

Mobile phase was freshly prepared, filtered and degassed prior

to the use. Ultrahydrogel 1000 (7.8 300 mm dimensions-

Waters) column was used and the flow rate was maintained at

0.6ml/min, while 20ml was injected into the column. The

absorbance of ibuprofen was measured at 280 nm.

2.5. Cell culture

Human lung epithelial carcinoma cell line (A549) was

obtained from Childrens Hospital of Michigan cell culture

facility and used for the cell uptake and drug activity studies.

Cells were grown in 75 mm2 culture flasks using RPMI 1640

(Invitrogen) cell culture medium supplemented with 10%

fetal calf serum (FCS-Invitrogen), pencillin (100 U/ml) and

streptomycin (100mg/ml) at 371C with 5% CO2 in an

incubator. The cells were subcultured every 48 h and harvested

from subconfluent cultures (6070%) using 0.05% trypsin

(Sigma, USA).

2.6. Flow cytometry analysis

A549 cells (seeded at 1.0105 cells/ml) were grown on

6015mm3 cell culture plates using RPMI 1640 cell culture

medium supplemented with 10% FCS, pencillin (100 U/ml)

and streptomycin (100mg/ml). When the cells were 60%

confluent, they were treated with FITC-labeled ibuprofen

(10mg/ml in ethanol) or FITC-labeled dendrimer conjugated

ibuprofen (equivalent to 10 mg/ml of ibuprofen in ethanol) for

5, 10, 15, 30, 45, 60, 120 and 240 min. Final concentration of

ethanol in the medium was 0.1% v/v and did not have any

effect on the cell. The cells were washed with phosphatebuffered saline (PBS, pH 7.4) trypsinized and centrifuged at

1500 rpm for 5 min to obtain a cell pellet. The cells were then

rinsed with PBS buffer, spun down twice, and resuspended in

PBS, and subsequently analyzed using a flow cytometer

(FACS caliber, Becton Dickinson) by counting 10,000 events.

The mean fluorescence intensity of the cells was calculated

using the histogram plot.

2.7. Cell supernatant analysis

The cell supernatant from the above study was removed at

times 0, 30, 60, 120, 240 and 360 min. Amount of FITC in the

supernatant was estimated by measuring the UV/Vis absor-bance of FITC at 496nm and quantified with a calibration

curve (using FITC-labeled drug conjugate) with appropriate

blank solution.

2.8. Fluorescence microscopy

The procedure for cell culture and drug treatment was

same as described in previous section. After treating with

the FITC-labeled drug conjugate for 4 h, the cells were

washed with phosphate-buffered saline (pH 7.4). A few drops

of the buffer and anti-fade reagent (Molecular Probes, USA)

was added before observing under the confocal microscope

(Zeiss LSM 310) using a magnification of 63X 1.2. The

emission and excitation wavelengths were 488 and 518nm

for FITC.

2.9. Pharmacological activity of PAMAM-G4-OH ibuprofen

conjugates

A549 lung epithelial cells (2.0105 cells/ml/well) were

seeded in 24 well plates and allowed to grow overnight in

RPMI 1640 medium supplemented with 10% FCS pencillin

(100 U/ml) and streptomycin (100mg/ml). When the cells

were 60% confluent, the medium was removed and washed

with serum-free medium (SFM). Each well was treated with

500 ml of SFM and prostaglandin (PGE2) secretion was

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P. Kolhe et al. / Biomaterials 27 (2006) 660669662


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induced by addition of 1.5mg of lipopolysaccharide (LPS)

and 1mg of interleukin (IL- 1b) to each well. After 30 min,

10mg of free ibuprofen and dendrimer conjugate (10mgm

equivalent of ibuprofen) in ethanol were added. Control

treatments with ethanol alone, PAMAM-G4-OH alone,

positive control with PGE2 induction, but no treatment and

negative control without any PGE2 induction were also

studied. The supernatant was removed at specific time intervalsof 30, 60 and 360 min and analyzed for PGE2 concentra-

tion using a commercial ELISA kit. Results were represented

as percent inhibition of PGE2 in comparison to positive

control.

3. Results and discussion

3.1. Chemistry and characterization

We have covalently conjugated the dendrimer to

ibuprofen by one-step synthesis reaction, through the

formation of an ester bond. For this, the selection of anappropriate dendrimer candidate for drug conjugation is

crucial. The higher generation cationic amine-termi-

nated dendrimers are sometimes cytotoxic when com-

pared to the neutral hydroxyl terminated dendrimers.

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Fig. 1. Schematic synthesis method for PAMAMdendrimeribuprofen conjugates.



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The appropriate dendrimers should have an adequate

number of reactive, surface end groups to conjugate the

drug ensuring optimal payload. We used G4-OH

PAMAM dendrimer which contains 64 hydroxyl groups

and is non-cytotoxic within the concentration range

used in the present study. The carboxylic acid group of

ibuprofen was conjugated with OH groups of PA-MAM-G4-OH dendrimer by using dicyclohexilcarbo-

diimide (DCC) as a coupling agent (Fig. 1). With one-

step reaction scheme, we expected to obtain high

payload of drug because of the multiple free surface

functional groups that are available on the periphery of

the dendrimer, and the high reactivity of the acid group

of ibuprofen.

The conjugates formed through this condensation

reaction were characterized using 1H NMR spectro-

scopy. The NMR spectrum of the PAMAM-G4-OH-

ibuprofen conjugate shows signals originating from bothPAMAM-G4-OH and ibuprofen (Fig. 2a). Multiplets

between d 2.0 to 3.7 ppm correspond to the presence of

985 protons of CH2 of PAMAM-G4-OH [21]. The two

doublets at 7.062 and 7.218 ppm correspond to aromatic

ARTICLE IN PRESS

Fig. 2. 1H NMR spectrum of PAMAMdendrimeribuprofen conjugate d-MeOH. The integration ratio for ibuprofen and dendrimer corresponds

to 58 molecules of ibuprofen per dendrimer. (b) GPC chromatogram of PAMAMdendrimer and the ibuprofendendrimer conjugate (inset).

PAMAM-G4-OH shows the retention time of 21.63 min. PAMAM-G4-OHibuprofen conjugate depicted earlier retention time of 16.83 min

signifying the formation of higher molecular weight conjugates.



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ring of ibuprofen and accounts for 4 protons per

ibuprofen molecule. The integration ratio of these

doublets to multiplets is 0.238, i.e., 234 (0.238985

234) protons of ibuprofen are present in conjugate

corresponding to the attachment of average number of

58 molecules of ibuprofen one molecule of PAMAM-

G4-OH. (Table 1). Approximate molecular weight hasbeen calculated for the conjugates based on this

information (Refer to Table 1) and based upon those

calculations the percentage of ibuprofen in the con-

jugates correspond to 47.44% by weight.

The polymeribuprofen conjugates were also evalu-

ated by GPC analysis. Fig. 2b shows the typical

chromatogram obtained for PAMAM-G4-OH and

PAMAM-G4-OHibuprofen conjugate. PAMAM-G4-

OH showed a retention time of 21.63 min. Although

PAMAM-G4-OH showed a strong peak at 21.63 min, a

small shoulder region is observed in the chromatogram.

The reason for the presence of shoulder peak is not clear

to us at this point. On the other hand, PAMAM-G4-

OHibuprofen conjugate exhibited a decrease in reten-

tion time to 16.83 min, signifying the presence of high

molecular weight conjugate. Pure ibuprofen showed a

retention time of 25 min (not shown). The absence of

any peak at 25 min signifies that PAMAM-G4-OHi-

buprofen conjugate sample was free of any unreacted

ibuprofen.1

It has been widely recognized that achieving high drug

payloads in hyperbranched polymers is a challenge. A

recent patent by Duncan et al. reported a drug payload

of 25% for cisplatin conjugated to dendrimers [22].

Earlier, investigators have conjugated typically 412 mo-lecules of drug or targeting agents to dendrimers

[8,23,24]. In the present study, we were able to conjugate

on an average 58 molecules of ibuprofen per dendrimer,

the highest reported in the literature to our knowledge.

These findings may suggest that the drug payload is

dependent on the choice of dendrimer, end function-

ality, and the reactivity of the functional group of the

drug used for conjugation. It appears that PAMAM-

G4-OH dendrimer along with ibuprofen as drug of

choice yielded high drug payload.

To summarize, we successfully synthesized conjugates

of dendrimers with ibuprofen, which yielded highpayloads through the formation of ester bond between

carboxyl group of ibuprofen and hydroxyl group of

dendrimer. The molecular weight distribution of these

conjugates was narrow based upon the qualitative

data from GPC. Previously, numerous polymer systems

have been used for drug-delivery application and poly

(2-hydroxypropyl) methacrylamide (HPMA) has shown

promise as a drug-delivery vehicle and currently in phase

I trial. Despite the promise shown by this system as far

as the drug action is concerned, producing monodis-

perse poly (HPMA) still remains a challenge [25,26].

Broad molecular weight distribution may lead to

variations in the pharmacokinetic behavior of the

drugpolymer conjugates [9]. Therefore, we believe that

this narrow molecular weight distribution of conjugatesmay lead to consistent in vivo results during animal

studies. High payload of drug molecules in conjugates

may be critical, as it can potentially increase the local

concentration of drug and resulting in higher therapeu-

tic efficacy with reduced systemic side effects.

3.2. Cell entry of conjugates

We investigated the cell entry dynamics of the high

payload drug conjugate by using a combination of flow

cytometry and UV/Vis spectroscopy. Dendrimeribu-

profen conjugates were fluorescently labeled with FITC

to evaluate its cellular uptake in A549 lung epithelial

cells. Using flow cytometry, it was found that the

dendrimerdrug conjugate entered cells rapidly. There

was significant fluorescence intensity increase within

15 min, corresponding to a conjugate uptake of $30%

(Figs. 3 and 4b). As evident from Fig. 3, the transport of

the conjugates into the cell increased with increasing

time. The results presented in Fig. 4 qualitatively show

the relative percentage of free or conjugated drug inside

and outside the cell. Extracellular drug levels were

monitored by measuring the UV absorbance of FITC

(in the conjugate) in the cell supernatant, while

intracellular drug levels were measured from the meanfluorescence intensity of the cells (due to FITC-labeled

ARTICLE IN PRESS

Fig. 3. Fluorescence activated cell sorter analysis of the cell entry

dynamics of ibuprofendendrimerFITC conjugate in A549 lung

epithelial cell line. The absorption intensity of FITC (FL1-H on x-

axis) is plotted against the number of cells (counts on y-axis). Key:

red0 min, green5 min, black15min, blue60 min and brown

240min.

1To substantiate, these findings, we attempted MALDI-TOF

analysis of the conjugates, but the acidic nature of the drug interfered

with the matrix, preventing clear data interpretation.



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drug or drugdendrimer conjugate) in a flow cytometer.

Both the techniques showed a reasonably good correla-

tion in following the transport of free as well as

dendrimer-conjugated ibuprofen. In order to rule out

the possibility that the cell entry dynamics of free

ibuprofen being dictated by the FITC label, we

monitored the cell entry dynamics of free ibuprofen bymeasuring the absorbance at 230 nm in a separate

experiment. The results were comparable (data not

shown) to results from FITC-labeled ibuprofen.

As shown in Fig. 4a and b, more than 40% of the free

ibuprofen and ibuprofendendrimer conjugate entered

cells within 60min. From the mean fluorescence

intensity, it was observed that there was no further

increase after 2 h (Fig. 4b). Although, both free and

dendrimer-conjugated ibuprofen shows similar profiles,

the conjugate may be expected to produce a high local

concentration of the drug in the cell based on the large

number of ibuprofen molecules attached to the den-

drimer molecule.

The cellular entry was also visualized by using

confocal microscopy. (Fig. 5ad) It is evident that the

FITC-labeled ibuprofen, dendrimer, and the dendri-

meribuprofen conjugates entered the cells and localiz-

ing mostly in the cytoplasm, while the nucleus appears

to be relatively free of the presence of any fluorescence

at this time scale. In contrast to the free ibuprofen, the

dendrimer-conjugated ibuprofen showed punctuated

distribution (the intracellular distribution pattern for

free dendrimer and dendrimeribuprofen conjugate

ARTICLE IN PRESS

0

20

40

60

80

100

120

0 50 100 150 200 250 300

%ofinitialconcentration

(b

yUVanalysis)

0

20

40

60

80

100

120

%of

finalfluoresence

inten

sity(FromFACS)

Extracellular

Intracellular

Extracellular

Intracellular

0

20

40

60

80

100

120

0 50 100 150 200 250 300

%o

finitialconcentration

(UVanalysis)

0

20

40

60

80

100

120

%offinalfluoresence

in

tensity(fromFACS)

Time in minutes

Time in minutes

(a)

(b)

Fig. 4. Decrease in FITC concentration in the cell supernatant (from

UV/visible spectroscopy shown with open squares on the left hand y-

axis) and increase in the FITC intensity inside the cell (calculated from

mean fluorescence intensity using flow cytometry and is shown with

closed squares on the right hand y-axis) for (a) IbuprofenFITC and

(b) IbuprofendendrimerFITC conjugate.

Fig. 5. Confocal fluorescence images of A549 cells after treatment with (A) Control (B) FITC-labeled G4OH dendrimer (C) FITC-labeled ibuprofen,

and (D) FITC-labeled ibuprofendendrimer conjugate after 4 h. The dendrimer, ibuprofen and conjugates appear to be localized in the cytoplasm

while the nucleus appears to be relatively free of the presence of any fluorescence at this time scale.



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were more diffuse in comparison to free ibuprofen)

(Figs. 5c and d), which is a characteristic feature of

endocytotic uptake This is a suggestive of an enhanced

intracellular uptake of the dendrimerdrug conjugate as

opposed to the free drug.

Cellular uptake of submicron particles could occur

through various processes such as phagocytosis, fluidphase pinocytosis or by receptor-mediated endocytosis

[27]. The lower molecular size cut-off described for

phagocytosis is 500 nm, which is much higher than those

of dendrimers (2030 nm), thus ruling out the possibility

of phagocytosis for cellular uptake of the dendrimer-

ibuprofen conjugate [28]. As there are no specific ligands

in this dendritic nanodevice, it is not expected to be

transported by receptor-mediated endocytosis. Epithe-

lial cells are known to possess anionic charge [29].

However, the PAMAM-G4-OH dendrimer cannot be

transported by ionic interactions, as they do not carry

any charge. Hence, it appears that the dendrimerdrug

conjugate is transported across the cell through fluid

phase endocytosis by non-specific interactions similar to

hyperbranched polymeribuprofen conjugates described

by Kolhe et al [30]. The results are further substantiated

by our recent findings that the cellular uptake of FITC-

labeled PAMAM G4-OH dendrimer was reduced in

presence of fluid phase endocytosis inhibitor (unpub-

lished data). Once inside the cell, the drugdendrimer

conjugate is expected to be transported by the endo-

somes which would then fuse with the acidic lysosomal

compartment [31]. This was evidenced by our recent

confocal microscopy studies, where, the FITC-labeled

PAMAM G4-OH dendrimer colocalized with thelysosomal marker in the cells (unpublished data). The

hydrolytic enzymes in the lysosomal compartment

would then cleave the polymerdrug conjugate [32]

and resulting in high intracellular concentration of the

drug. On the other hand, free ibuprofen due to its small

size would be transported by passive diffusion where the

intracellular concentrations of the drug would be

dictated by the concentration gradient.

3.3. Anti-inflammatory activity of ibuprofen dendrimer

conjugate

Ibuprofen is a non-steroidal anti-inflammatory drug

(NSAID), a derivative of propionic acid and is widely

used as an analgesic. Ibuprofen alleviates pain by

inhibiting the synthesis of prostaglandin. Prostaglandins

are integral in the bodys control of vasoconstriction

and inflammation, so reducing prostaglandin synthesis

reduces inflammation and the perceived pain associated

with the inflamed tissue. The mode of action of

ibuprofen involves the acetylation of cyclooxygenase-2

(COX-2) which blocks access and egress to/from the

active site, inhibiting the production of prostaglandin

[33]. We evaluated the efficiency of the dendrimeribu-

profen conjugate to suppress COX-2 by measuring the

prostaglandin (PGE2) in the cell supernatant. Free

ibuprofen did not inhibit the prostaglandin release from

A549 cells after 30 min incubation, while the dendri-

meribuprofen conjugate showed a significant inhibition

within the same time period (po0:05). However, at 60

and 360 min, both the free and conjugated ibuprofen

inhibited prostaglandin release to the same extent

(p40:05) (Fig. 6). Neither blank dendrimer nor the

solvent showed any suppression of PGE2 synthesis. The

above results imply that conjugates rapidly enter the cell

and produce the desired pharmacological action at the

target site in the cytosol. Though, a moderate fraction offree ibuprofen enters the cells within 30 min, it may not

achieve a sufficient concentration to elicit a rapid

pharmacological response. On the other hand, dendri-

meribuprofen conjugate, achieves a high local concen-

tration in the cell due to its high drug payload. At this

point, it is unclear whether the ibuprofen is released

from the nanomaterial inside the cell or if the drug is

effective even in the conjugated form. However, once the

device enters inside the cell, it is conceivable that the

acidic pH and the enzymes in the endosomes would

hydrolyze the ester bond in the conjugate [27], thereby

releasing the free drug in the cytosol to suppressprostaglandin synthesis. Further studies are underway

to study the stability of dendrimerdrug conjugate at

various pH and in presence of the enzymes to explore

this phenomenon. However, it has been reported in the

literature that the ester linkage of dendrimer conjugated

to the chemotherapeutic drugs, such as methotrexate

(MTX) typically have lower toxicity and higher tumor

cell killing efficacy than free drug. The authors

concluded that the dendrimerMTX conjugate enters

the cells efficiently and the ester bond is hydrolyzed at

the low pH found in the endosome, thereby releasing

free MTX [24]. Assuming comparable hydrolysis of ester

ARTICLE IN PRESS

0

20

40

60

80

100

120

30 60 360

PercentinhibitionofPGE2

Ibuprofen

G4-Ibuprofen conjugate

Time in minutes

Fig. 6. Percent inhibition of prostaglandin (PGE2) for free ibuprofen

and PAMAM-G4-OHibuprofen conjugate as a function of treatment

time at 30, 60 and 360min (average of four measurements with error

bars). Blank dendrimer and solvent did not show any inhibition of

prostaglandin release. The G4ibuprofen conjugate shows inhibition

of PGE2 release as early as 30 min.



9/10

bond inside the cells, our high payload drug conjugates

would show a significantly more rapid therapeutic effect

than the free drug. Pharmacodynamic studies are

ongoing in vivo in rats to investigate the superiority of

dendrimeribuprofen conjugate over free ibuprofen.

The conjugation of the drug to the dendrimer

conjugation may enable a high local drug concentrationat the target site and potentially overcome the systemic

adverse effects of free ibuprofen. For parenteral admin-

istration, nanomaterials would provide superior blood

stability and would enable sustained drug levels leading

to reduced dose frequencies. Therefore, these dendritic

polymers will help deliver a larger payload of the drug

faster while improving circulation times significantly.

This will decrease the dosage required to achieve the

same effect and more efficient delivery of the drug will

lead to a greater therapeutic efficacy while decreasing

the incidence of side effects of these drugs.

4. Conclusions

Drug conjugation, cellular transport, and cellular

therapeutic activity of dendrimer-based drug-delivery

vehicles are investigated. This study demonstrates the

potential of achieving a high drug payload using

PAMAM G4-OH dendrimer, through a DCC coupling

reaction, resulting in the formation of ester bond

between the dendrimer and the drug. Approximately

58 molecules of the drug were conjugated to one

dendrimer molecule containing 64 end groups. The

dendrimerdrugFITC conjugate appears to enter A549

lung epithelial cancer cell lines rapidly, and localizes

predominantly in the cytoplasm. At short time scales,

the conjugated drug appears to show superior PGE2suppression, suggesting higher activity for the conju-

gated drug. Drug dendrimer conjugates provides a mode

for intracellular drug delivery achieving a high local

concentration of the drug as opposed to the simple

diffusion of small molecular weight ibuprofen. The high

drug payload dendritic nanodevices translate into rapid

pharmacological response with improved efficacy. Fu-

ture studies are warranted to evaluate the pharmacoki-

netic and pharmacodynamic aspects of these nano-

materials in animal models.

Acknowledgements

This research work was funded by National Science

Foundation through DMR Grant # 9876221, Childrens

Research Center of Michigan (Childrens Hospital of

Michigan), WSU research enhancement funding and

NIH- Pediatric Pharmacologic research unit supple-

mental funding (NIH 3U01HD-37261-04SI). We would

like to thank Prof. David Bassett for the help with

FACS measurements.

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