Pegylated dendrimer and its effect in

The 5-FU encapsulated pegylated dendrimer exhibited a significant decrement in volume of the tumors which was generated by MCF-7 cancer cells.. In this article, we prepared the pegylated

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Biomedical Nanotechnology Vol 9, 1–8, 2012

Pegylated Dendrimer and Its Effect in Fluorouracil Loading and Release for Enhancing Antitumor Activity

Tu Uyen Ly1  †, Ngoc Quyen Tran1  3 †, Thi Kim Dung Hoang1, Kim Ngoc Phan2,

Hai Nhung Truong2, and Cuu Khoa Nguyen1  3 ∗

1Institute of Chemical Technology, Vietnam Academy of Science and Technology, HCMC 70000, Vietnam

2Stem Cell Research and Application Laboratory, University of Science HoChiMinh City, HCMC 70000, Vietnam

3Institute of Applied Materials Science, Vietnam Academy of Science and Technology, HCMC 70000, Vietnam

Dendrimer, a new class of hyper-branched polymer with predetermined molecular weight, is being

received much attention in nano biomedical applications such as anticancer drug delivery, gene

therapy, disease diagnosis and etc In this study, polyamidoamine (PAMAM)-based dendrimer

gen-eration 3.0 (G 3.0) was synthesized and subsequently pegylated Obtained results showed that

pegylation degree of the dendrimer was around 31% for its external amine groups TEM image

of the pegylated dendrimer exhibited spherical shape and nano sizes ranging from 30 to 40 nm

The fluorouracil (5-FU)-loaded pegylated dendrimer showed a slow release profile of the drug

In vitro study, at the primary screening concentration of 100 g/mL, the PAMAM dendrimer

pre-sented higher toxicity in MCF-7 cells as compared to its pegylated counterpart Meanwhile, the

(5-FU)-loaded pegylated dendrimer exhibited the antiproliferative activity against the cell line with

the IC50 of 992 ± 019 g/mL In vivo tumor xenograft study, we succeeded in generating MCF-7

cells-derived cancer tumors on mice that was well-confirmed by using flow cytometer assay The

5-FU encapsulated pegylated dendrimer exhibited a significant decrement in volume of the tumors

which was generated by MCF-7 cancer cells

Keywords: Fluorouracil, Pegylated Dendrimer, Anticancer, Drug Delivery

1 INTRODUCTION

Dendrimer is a family of hyper-branched polymers that

was introduced in 1980’s by Donald A Tomalia

Synthe-sis of the dendrimers involves a molecular growth process

which occurs through step-wise polyester formation or

Michael addition and amidation of multifunctional groups

to issue layer of branches around the core The

reac-tions result in producing a precisely defined chemical

structure of PAMAM or polyester dendrimer.1 2 In the

family, PAMAM is one of the most studied dendritic

poly-mers For the high-generation dendrimers (G3, G4, G5  ),

these structures possess internal cavities which can be

ultilized as a novel nanocarrier for anticancer drug delivery

because drugs can be encapsulated via non-covalent

inter-actions Moreover, its externally exposed amine or

car-boxylic groups could be decorated with targeting or/and

∗ Author to whom correspondence should be addressed.

†These authors contributed equally to the work.

drug molecules.3 4Several reports indicated that anticancer

drugs (camptothecin, 6-mercaptopurin, methotrexate, adri-amycin, 5-fluorouracil, and paclitaxel) were encapsu-lated into the PAMAM dendrimer exhibiting a significant enhancement of its water solubility, storage stability, and anti-tumor activity.5–10

However, there are a few disadvantages accompanied with PAMAM dendrimer drug-delivery system includ-ing hemolytic toxicity and cell lysis due to a strong interaction of the positively charged dendrimer and the negatively charged cell membrane resulting in mem-brane disruption.9 11–13 Like other cationic polymers such

as polylysine and poly(ethyleneimine), these disadvan-tages have been solved by conjugating biocompatible and hydrophilic polymers into amine group-terminated dendrimer Up to now, polyethylene glycol (PEG) has been one of the best choices for the research PEG is well-known that is highly water soluble, nontoxic and nonimmunogenic The pegylation can help in reducing

toxicity by preventing the contact between terminal

protonated amine groups with cell membranes, resulting

in improving their biocompatibility The conjugation may

lead to increase in the inner cavity space of dendrimers

that contribute to increment of drug-loading capacity.14

Moreover, pegylation of the drug nanocarriers can increase

the residence time of the drug in blood circulation by its

stealth properties in the blood plasma These improving

effects of pegylated nanocarriers were well-confirmed in

several studies, both in vitro and in vivo.8 9 14 15 Despite

this, little effort has been made to evaluate treatment

effi-cacy on implanted tumor tissue

In this article, we prepared the pegylated PAMAM

den-drimer G3.0 for loading 5-fluorouracil anticancer drug

The in vitro and in vivo effectiveness against human breast

cancer MCF-7 cell line of this complex was investigated

using Sulforhodamine B colorimetric assay and xenograft

technique, respectively

2 MATERIALS AND METHODS

2.1 Materials

Ethylenediamine (EDA), methyl methacrylate, and

5-fluorouracil were purchased from Merck Chemicals

Monomethoxy polyethylene glycol 5000 (MPEG-5000)

was obtained from Sigma-Aldrich Co.p-nitrophenyl

chlo-roformate (NPC) was purchased from Acros Organics

Fig 1 Synthetic scheme of Pegylated PAMAM dendrimer G3.0.

PAMAM dendrimers G3.0 were prepared in Organic Chemistry and Polymer laboratory16(Institute of Chemical Technology, Vietnam Academy of Science and Tech-nology) following the procedure reported by Tomalia1

Regenerated Cellulose MWCO 3500-5000D and Cellulose Ester MWCO 10000D dialysis bags were purchased from Spectrum Laboratories Inc All other chemicals were used without further modification

2.2 Synthesis of Pegylated PAMAM Dendrimer G3.0

Activated MPEG-5000 was prepared similarly to the method described by Tran et al Five grams of dried MPEG

5000 (1.0 mmol) was completely dissolved in dimethyl-formamide at 40 C and then reacted with p-nitrophenyl

chloroformate (2.0 mmol) in the presence of triethylene amine under nitrogen atmosphere The mixture was stirred overnight The product was precipitated in excess diethyl ether to obtain a white powder of the activated MPEG The product was then dried and used further synthesis.17

A mixture of PAMAM G3.0 dendrimer (155.0 mg,

896.0mol) in 30 mL of DMF was stirred under nitrogen

atmosphere for 48 h (Fig 1) The crude product was dialyzed (MWCO 10,000D) against water under strict sink conditions in 48 h The product was then lyophilized and used for drug loading preparation

2.3 Drug Loading and In Vitro Release Evaluation

5-FU was loaded in pegylated PAMAM dendrimers

fol-lowing the equilibrium dialysis method reported earlier.9

The pegylated PAMAM dendrimer G3.0 (428.3 mg,

con-taining 100 molar times of 5-FU (97.0 mg, 746mol) The

mixed solution was incubated under slow stirring (50 rpm)

for 24 h This solution was twice dialyzed under strict

sink conditions in 20 min to remove free drug from the

formulation, which was then estimated

spectrophotometri-cally (265.5 nm) to determine indirectly the amount of

drug loaded within the system Optimal drug loading in the

nanocarrier was determined on the same method Briefly,

pegylated dendrimer was dissolved in water at 0.75 mM

polymer concentration And then, an excess 5-FU was

added to the pegylated dendrimer solution under slow

stir-ring (50 rpm) for 24 h The mixture was centrifugated

(5000 rpm) to remove the amount of insoluble 5-FU

Fol-lowing the above method, the optimal drug loading could

be determined within the system The dialyzed formulation

was lyophilized and used for further studies The

drug-loading (DL%) and entrapment efficiency (EE%) of 5-FU

in pegylated dendrimer were calculated from the following

equations.18 19

DL%=Weight of 5-FU in nanocarrier

Weight of 5-FU in nanocarrier× 100%

EE%=Weight of 5-FU in nanocarrier

Weight of feeding 5-FU × 100%

For in vitro release study, the 5-FU-loaded pegylated

den-drimer (260.6 mg) and 10 mL deionized water were added

to dialyzer membrane (MWCO 3,500D) The aqueous

con-taining membrane was dialyzed against 1000 mL

deion-ized water At a predetermined time interval, 10 mL of

dialyzed solution was drawn to determine 5-FU release

by absorbance measurement at wavelength 265.5 nm and

another 10 mL of deionized water was added to the

dia-lyzed solution to compensate for the withdrawn volume

The similar concentration of dendritic polymer solution

without drug loading was dialyzed in the same condition

to serve as control

2.4 Cytotoxicity Assays

The cell proliferation was measured using Sulforhodamine

B (SRB) colorimetric assay.20The inhibition capability of

cell growth of PAMAM, pegylated PAMAM, free 5-FU

and pegylated PAMAM dendrimer 5-FU complex was

esti-mated at the screening concentration of 100g/mL

MCF-7 cells (Frederick, MD, U.S.A) were seeded at a density

of 104cells per well in 96 well plates and allowed to grow

in culture medium (DMEM/F12 containing 10% FBS,

1% antibiotics and 5% CO2 atmosphere) overnight These

wells were then incubated with the medium containing

tested compound for 48 h A negative control was culture medium Blank sample was culture medium containing compound but without cells After the incubation period, the cells were fixed with 50% (wt/vol) trichloroacetic acid and stained with 0.2% (wt/vol) SRB for 20 min The excess dye was removed by washing repeatedly with 1% (vol/vol) acetic acid Finally, the protein-bound dye was dissolved in 10 mM Tris-base solution for optical density (OD) determination at 492 and 620 nm using a Multiskan Ascent Reader (Thermo Electron Corporation) The OD at certain wavelength was defined as the mean absorbance of tested wells minus the blank value The OD value of each sample was subsequently calculated as the

OD at 492 nm subtracting from the background measure-ment at 620 nm The percentages of cell growth inhibition were calculated using the formula below

For IC50 estimation, cell viability was analyzed

at different material concentrations using sulforho-damine colorimetric assay Based on the dose–response curve between the compound concentration and growth inhibition percent, the IC50 values were subsequently determined using regression analysis Experiments were performed in triplicates for each compound and each experiment was carried out at least twice The values were expressed as means± standard deviation (STD)

The study was conducted at Stem Cell Research and Application Laboratory, University of Science - Vietnam National University HCM City Briefly, twenty Swiss mice

of similar weights and sizes were used in this experi-ment Before injecting MCF-7 human breast cancer cells, the mice were administered immunosuppressive drugs (20 mg/kg of Busulfan and 200 mg/kg of Cyclophos-phamide per day for five days) to suppress the immune system The tumor bearing mice were created by sub-cutaneous injection of MCF-7 into mouse’s thigh at a same cell density (107 cells/animal) After two weeks, the animals (n = 12) that their tumors reached required and

less changed volumes were divided into three individual groups The first group served as control, the second group was given a dose of 10 mg/kg 5-FU per day, the other was given a dose of pegylated PAMAM dendrimer-drug complex with an equivalent amount of 10 mg/kg 5-FU per day The animals were treated in 15 days and the changes

of the tumor volumes were recorded during the treatment The existence of MCF-7 cells in tumors was confirmed by flow cytometry using anti-HLA monoclonal antibody All experimental procedures and manipulations were approved

by our Institutional Ethical Committee (Laboratory of Stem cell Research and Application, University of Science, VNU-HCM, VN)

2.6 Characterizations

Nuclear magnetic resonance (NMR) data was collected

using CDCl3 as solvent on a Bruker AC 500 MHz

spec-trometer The average molecular weights of pegylated

polymer was calculated from Gel Permeation

Chromatog-raphy (GPC) technique using Agilent 1100-GPC system

Deionized water was used as an eluent at a flow rate of

1 mL/min through a Ultrahydrogel column Peak

analy-sis was performed basing on a universal calibration curve

generated by a pulullan polysaccharide standard of

nar-row polydispersity For Transmission Electron Microscopy,

pegylated dendrimer was dissolved in methanol at a certain

concentration, placed on 300 mesh carbon-coated copper

grid, and then air-dried for several hours TEM images

(TEM) were obtained at 100 kV with a JEM-1400 (JEOL)

with magnifications up to 100,000×

3 RESULTS AND DISCUSSION

3.1 Synthesis of Pegylated PAMAM G3.0 Dendrimer

PEGylation was a common method to reduce

immuno-genicity of proteins or drug nanocarriers The hydroxyl

group of MPEG chain could be activated byp-nitrophenyl

chloroformate (NPC) The NPC-activated MPEG could be

well confirmed by1H NMR, in which there were two

dou-blets at 8.29–8.27 ppm and 7.41–7.39 ppm corresponding

to the protons of the phenyl The activation was also

well-proved by the chemical shift of the protons (Hc) of the

ter-minal CH2 group that was originally attached to hydroxyl

group towards lower frequency region, represented by a

multiplet at 4.45–4.44 ppm (Fig 2)

Calculation from the integral ratio of the proton signals

of the benzene ring (in the p-nitrophenyl group) and the

signal of the protons of the terminal methoxy group in

MPEG chain (at 3.40 ppm) showed that the conversion

rate of MPEG to its activated form was nearly complete

with a yield of about 92%.21

The activated MPEG was utilized for pegylation of

PAMAM dendrimer (Fig 1) As conjugated to PAMAM,

the signal of MPEG methylene protons next to the

pre-viously activated group shifted from 4.45 to 4.18 ppm

Fig 2 1 H NMR spectrum of the NPC-activated MPEG.

Fig 3 1 H NMR spectrum of the pegylated PAMAM dendrimer G3.0.

(Fig 3) Besides appearance of typical peaks for MPEG methylene and methyl protons, PAMAM methylene pro-tons could be also presented in the Figure 3

The obtained GPC result demonstrated (Fig 4) that the PEGylation degree was about 30% as estimated between Mw of PAMAM (6,909 g/mol) and its pegylation

(57,800 g/mol; PDI= 14) Ten amine groups had been

pegylated among total thirty two amine groups It was said that the pegylated product could help in reducing toxic-ity by preventing the contact between terminal protonated amine groups with cell membranes, resulting in improv-ing its biocompatibility Moreover, it can contribute to the stealth properties in the blood plasma and improve the specificity of the pharmacodynamic action.22 23

The pegylation of dendrimer could be well-defined by TEM in which its morphology is spherical shaped and diameter ranging from 30 nm to 40 nm (Fig 5) There

is a significant size increment of the pegylated PAMAM dendrimer as compared to the original PAMAM dendrimer G3.0 (diameter under 5 nm; data not shown here) Structural characterization of the pegylated dendrimer showed an increment of nanoparticles size that can lead to increase in the inner cavity space of the dendrimers and contribute to increment of drug-loading capacity

Fig 4 GPC result of the pegylated PAMAM dendrimer G3.0.

Fig 5 TEM image of the pegylated PAMAM dendrimer G3.0.

3.2 Drug Loading and In Vitro Release

The drug formulation was prepared by incubating the

pegylated PAMAM dendrimer with a saturated 5-FU

solu-tion The amount of drug that had been loaded was

calcu-lated indirectly from the amount of unbound drug which

was determined spectrophotometrically at the wavelength

of 265.5 nm In this study, the loading efficiency was

about 30% (as shown in Table I), calculating

approxi-mately 30 drug molecules that were found to be

encapsu-lated within each pegyencapsu-lated PAMAM dendrimer molecule

structure Moreover, the optimal drug loading was

deter-mined around 35% as shown in Table I The table also

showed that entrapment efficiency of 5-FU in the pegylated

dendrimer didn’t significantly increase as used of excess

5-FU According to drug loading (DL%) and entrapment

efficiency (EE), we thought that use of pegylated

den-drimer for loading a saturated 5-FU solution may be more

effect than loading 5-FU saturated in the pegylated

den-drimer solution

Signal of 5-FU proton (7.61–7.62 ppm) could be

observed in 1H NMR spectra of drug loaded dendrimer

after the free drug was removed using dialysis

mem-brane (data not shown) Therefore, this could be concluded

that 5-FU was simply physically entrapped inside

pegy-lated PAMAM dendrimer cavities Release profile of the

encapsulated drug molecules under strict sink conditions

is shown in Figure 6

The release profile shows an initial burst release of 40%

5-FU from the drug-loaded dendrimer within the first hour

of the experiment After that time, the drug slowly releases

Table I Drug-loading (DL%) and entrapment efficiency (EE%) in

pegylated dendrimer (n = 3).

PAMAM + saturated 5-FU solution 30.15± 1.27 5.54± 0.32

Saturated 5-FU in PAMAM solution 34.75 ± 3.71 10.65± 0.78

Fig 6 Release profile of 5-FU from the drug-loaded dendrimer.

from the system and reaches to more than 84% released at

24 hours This behavior is very significant to prolong drug bioavailability because 5-FU anticancer drug was reported

to have a short remaining time in blood circulation The drug can be excreted or metabolized 95% out of blood plasma after one hour of administration.9 24

3.3 Cytotoxicity Assay and IC50

The antiproliferative activities of PAMAM, pegylated PAMAM, free 5-FU drug and 5-FU-loaded pegylated PAMAM on MCF-7 cells were expressed indirectly via the cellular protein content that could electrostatically bound

to sulforhodamine B dye molecules in the assay The obtained results showed cytotoxicity of PAMAM and its pegylated derivative were negligible at the experimental condition (shown in Table II) However, the result obvi-ously showed pegylation can reduce the cytotoxic ability

of PAMAM Meanwhile, the percentages of cell growth inhibition of the two free and entrapped 5-FU samples at the primary screening concentration were too high There-fore, IC50 estimation assays were conducted to assess accurately the cytotoxicity The IC50 values were deter-mined at 105 ± 019 g/mL and 992 ± 019 g/mL for

5-FU free drug and entrapped 5-FU samples, respectively

It was not surprising that the free drug seemed to be nine

Table II. In vitro cytotoxicity of PAMAM, pegylated PAMAM, free

5-FU drug and 5-FU encapsulated pegylated PAMAM on MCF-7 cancer cell.

Sample Concentration Antiproliferative activity

cell growth Pegylated PAMAM 100g/ mL Inhibited 6.36 ± 1.42%

cell growth Free 5-FU 1.05± 0.19 g/ mL Inhibited 50% cell

growth 5-FU encapsulated 9.92± 0.19 g/ mL Inhibited 50% cell

growth pegylated PAMAM

times more effective than the encapsulated drug because

the drug content in the pegylated carrier was quite low The

total thirty drug molecules per pegylated PAMAM

den-drimer molecule werewas only equivalent to around 6.5%

(wt/wt) Therefore, it could be concluded that 5-FU loaded

inside the pegylated dendrimer still maintained an

signifi-cantly antiproliferative activity on the MCF-7 cancer cell

The result could be attractive to studies on drug

nanocar-rier and its utilization for many toxic anticancer drugs

Before using in vivo experiments, the cell suspension

was analyzed with flow cytometer which confirmed being

MCF-7 breast cancer cell using HLA monoclonal

anti-body Figures 7(a) and (b) showed that the cells suspension

was predominantly MCF-7 cell with 97.51% of

popula-tion The cells in the generated cancer tumor via xenograft

assay were re-collected and determined integrin

expres-sion again by flow cytometer to confirm that they were

predominantly MCF-7 cells (Fig 7(c)) After two week

of implantation, the MCF-7 cells easily generated cancer

tumors on each mouse’s thigh (Fig 8(a)) Those cancer

tumors were then exploited to evaluate tumor-killing

effi-cacy of the pegylated dendrimer loading 5-FU To remove

the errors caused by the inconsistence in initial tumor

vol-umes, and the variation in physiological reponses from the

treatment, the in vivo efficacy was evaluated on the

aver-age percentaver-age decrease in tumor volume The obtained

results showed that the tumor volume on mice decreased

as treated 5-FU and the 5-FU loaded in the pegylated

den-drimer (Figs 8(b and c))

In comparison among the three studied groups, group

treated 5-FU encapsulated pegylated PAMAM dendrimer

gave the best treatment results The tumor volumes were

decreased gradually over the time, and the maximum

decreasing percentage was found after 15 days and it

was 82.87% that was far exceeding result of the free

5-FU treated group, which was decrement in tumor

vol-umes being 42.29% Meanwhile, the control group had

increment in tumor volumes approximately 21.58% The

decrement of tumor volumes is a result from anticancer

activity of 5-FU, a commercial anticancer drug that is

administrated for long-term treatment of cancer patients

The tumor-killing efficacy in the three groups over the

study time is illustrated in Figure 8(d) 5-FU encapsulated

the pegylated PAMAM dendrimer and showed a higher

efficacy of tumor killing in comparison with free 5-FU

treatment This could be explained that 5-FU encapsulated

in pegylated PAMAM dendrimer prolonged drug

bioavail-ability due to a slow drug release It is well-known that

5-FU anticancer drug being a short remaining time in

blood circulation, 95% out of blood plasma after one hour

of administration.9 22 Figure 8(c) also shows that a high

standard deviation can be seen in all studied groups This

Fig 7 Expression of HLA-DR on MCF-7 cells Flow cytometer analy-sis of the MCF-7 cells at SSC versus FSC histogram (a), HLA-DR FITC versus count (b) and the tumor-isolated MCF-7 cells were determined

by flow cytometer using anti-HLA antibody (c) SSC: Side Scatter, FSC: Forward Scatter.

Fig 8 Results of xenograft assay: mouse with generated cancer tumor

(a), mouse with tumor after treated with the 5-FU loaded dendrimer (b)

and decrement percentage of tumor volumes (c) (n = 4 ± SD).

is due to an initial difference in tumor volumes among

mice that could be improved by increment in amount of the

studied mice To further clarify the tumor-killing effect of

the drug-loaded pegylated dendrimer, administrative

treat-ments at various dose levels and time interval for the

treated doses as well as a larger amount of the studied

mice are going on study

4 CONCLUSION

The pegylated PAMAM dendrimer was prepared and

well-defined in structure Drug-loaded dendrimer was

formu-lated, wherein the 5-FU drug molecules were physically

entrapped in the cavities of the structure of pegylated

PAMAM dendrimer The drug-loaded pegylated dendrimer

maintained subtantial antiproliferative activity of free drug

on the MCF-7 cell line in vitro, and furthermore showed a

significant improvement in the anti-tumor activity as com-pared to controls without drug and with free drug treatment

in vivo The obtained results may contribute to further

studies and applications in killing cancer cell of tumors by using the nanocarrier

Acknowledgments: This work was financially sup-ported by Material Science and Technology project of Vietnam Academy of Science and Technology

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Received: 1 December 2011 Revised/Accepted: 25 June 2012