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Biomaterials 27 (2006) 660669 www.elsevier.com/locate/biomaterials

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,
Department of Chemical Engineering and Material Science, and Biomedical Engineering, Wayne State University, 5050, Anthony Wayne Drive, Detroit, MI-48202, USA b Department of Pediatrics (Critical Care Medicine), Childrens Hospital of Michigan, Wayne State University, Detroit, MI-48201, USA Received 28 February 2005; accepted 20 June 2005 Available online 27 July 2005
a

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 uoroisothiocynate (FITC)-labeled dendrimerdrug conjugate was evaluated in vitro by using human lung epithelial carcinoma A549 cells by ow 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. Signicant 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 efcacy by enhanced cellular delivery, and may produce a rapid pharmacological response. These dendrimerdrug conjugates can potentially be further modied 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 temporal control 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 fundamental level, function and structure necessary for life,
Corresponding author. Tel.: +1 313 577 3879; fax: +1 313 577 3810. E-mail address: rkannan@chem1.eng.wayne.edu (R.M. Kannan).

result from specic molecular structures and shapes in the nanometer scale. Hence, molecular level strategies to target, deliver, detect, diagnose and treat diseases could be more therapeutically efcacious compared to a systemic 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-dened globular shape ($20 nm) and multivalency [3,4]. These salient

0142-9612/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2005.06.007

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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 modify dendrimers 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 targetability [15,16]. Although signicant 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 of therapeutic areas where the target site is intracellular, such as pain management and inammation. The main issues associated with the use of dendrimers for drug delivery include, overcoming cytotoxicity especially in the case of cationic dendrimers, improving drug payload, 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 G4OH dendritic nanomaterials to deliver the drug intracellularly. Studies have shown the potential to target chemotherapeutic agents to tumor cells mostly with

cationic dendrimers [8,9,18]. However, neutral dendrimers 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-terminated 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 synthesis and evaluation of OH-terminated PAMAM dendrimeribuprofen conjugates with a high drug payload for enhanced cellular delivery. Ibuprofen is a nonsteroidal anti-inammatory drug (NSAID), which shows side effects such as renal dysfunction and gastrointestinal hemorrhage when delivered by conventional drug-delivery systems. Improving the efcacy 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 dendrimer (average molecular weight $14,279 Da) 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 (IL1b) 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

Table 1 Molecular properties of PAMAM-G-4-OH-terminated dendrimer and ibuprofen conjugation ratios Avg. Mw (g/mol) 14,279a 206 No. of end groups % Mole of ibuprofen in conjugate 98
b

% Wt. of ibuprofen in conjugate 47.44

Average No. of ibuprofen in conjugate 58

Average Mw with ibuprofen in the conjugates (g/mol) 25,183

PAMAM-G4-OH Ibuprofen
a b

64 1

Reported by Tomalia et al. (1990). Estimated by 1H NMR integration method.

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(DMF) were purchased from Fischer Scientic. 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 was calculated on the basis of molecular weight and number of end groups of the dendrimer). To this solution, DCC (1.5 g, 7.28 mM) was added as a coupling agent and the reaction was stirred continuously for 3 days at room temperature. The reaction mixture was ltered 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 temperature to obtain dendrimeribuprofen conjugates. Repurication 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 1H NMR. 2.3. Synthesis of PAMAM-G4-OH ibuprofen FITC conjugates FITC (0.02 g, 0.051 mM) was rst conjugated with glutaric acid (0.067 g, 0.051 mM) to form a carboxyl-terminated FITC using DCC (0.09 mg) as a coupling agent. Further, PAMAMG4ibuprofen conjugates (0.5 g, 0.0019 mM) and FITCglutaric acid moiety (0.078 g, 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 room temperature and ltered to remove N, N0 -dicyclohexylurea. The solution was ltered and dialyzed against DMSO for 24 h to remove unreacted FITCglutaric acid and DCC. The contents inside the dialysis membrane were removed and further puried with acetone to remove free FITCglutaric acid. Absence of free FITC in the conjugate was veried 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 instrument equipped with manual injector and UV detector interfaced to Breeze software. The mobile phase used was 0.05 M NaHCO3/ 0.1 M NaOH/deionized water (50:23:27) with a pH of 11. Mobile phase was freshly prepared, ltered and degassed prior to the use. Ultrahydrogel 1000 (7.8 300 mm dimensionsWaters) column was used and the ow rate was maintained at 0.6 ml/min, while 20 ml 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 asks using RPMI 1640 (Invitrogen) cell culture medium supplemented with 10% fetal calf serum (FCS-Invitrogen), pencillin (100 U/ml) and streptomycin (100 mg/ml) at 37 1C with 5% CO2 in an incubator. The cells were subcultured every 48 h and harvested from subconuent cultures (6070%) using 0.05% trypsin (Sigma, USA). 2.6. Flow cytometry analysis A549 cells (seeded at 1.0 105 cells/ml) were grown on 60 15 mm3 cell culture plates using RPMI 1640 cell culture medium supplemented with 10% FCS, pencillin (100 U/ml) and streptomycin (100 mg/ml). When the cells were 60% conuent, they were treated with FITC-labeled ibuprofen (10 mg/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 phosphate buffered 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 ow cytometer (FACS caliber, Becton Dickinson) by counting 10,000 events. The mean uorescence 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 absorbance of FITC at 496 nm and quantied 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 magnication of 63X 1.2. The emission and excitation wavelengths were 488 and 518 nm for FITC. 2.9. Pharmacological activity of PAMAM-G4-OH ibuprofen conjugates A549 lung epithelial cells (2.0 105 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 (100 mg/ml). When the cells were 60% conuent, 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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induced by addition of 1.5 mg of lipopolysaccharide (LPS) and 1 mg of interleukin (IL- 1b) to each well. After 30 min, 10 mg of free ibuprofen and dendrimer conjugate (10 mgm 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 specic time intervals of 30, 60 and 360 min and analyzed for PGE2 concentration 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 an appropriate dendrimer candidate for drug conjugation is crucial. The higher generation cationic amine-terminated dendrimers are sometimes cytotoxic when compared to the neutral hydroxyl terminated dendrimers.

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 PAMAM-G4-OH dendrimer by using dicyclohexilcarbodiimide (DCC) as a coupling agent (Fig. 1). With onestep 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 spectroscopy. The NMR spectrum of the PAMAM-G4-OHibuprofen conjugate shows signals originating from both PAMAM-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

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 (0.238 985 234) protons of ibuprofen are present in conjugate corresponding to the attachment of average number of 58 molecules of ibuprofen one molecule of PAMAMG4-OH. (Table 1). Approximate molecular weight has been calculated for the conjugates based on this information (Refer to Table 1) and based upon those calculations the percentage of ibuprofen in the conjugates correspond to 47.44% by weight. The polymeribuprofen conjugates were also evaluated by GPC analysis. Fig. 2b shows the typical chromatogram obtained for PAMAM-G4-OH and PAMAM-G4-OHibuprofen conjugate. PAMAM-G4OH 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-G4OHibuprofen conjugate exhibited a decrease in retention 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 signies that PAMAM-G4-OHibuprofen 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 molecules 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 ndings may suggest that the drug payload is dependent on the choice of dendrimer, end functionality, and the reactivity of the functional group of the drug used for conjugation. It appears that PAMAMG4-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 high payloads 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
1 To substantiate, these ndings, we attempted MALDI-TOF analysis of the conjugates, but the acidic nature of the drug interfered with the matrix, preventing clear data interpretation.

I trial. Despite the promise shown by this system as far as the drug action is concerned, producing monodisperse 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 conjugates may 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 therapeutic efcacy 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 ow cytometry and UV/Vis spectroscopy. Dendrimeribuprofen conjugates were uorescently labeled with FITC to evaluate its cellular uptake in A549 lung epithelial cells. Using ow cytometry, it was found that the dendrimerdrug conjugate entered cells rapidly. There was signicant uorescence 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 mean uorescence intensity of the cells (due to FITC-labeled

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 xaxis) is plotted against the number of cells (counts on y-axis). Key: red0 min, green5 min, black15 min, blue60 min and brown 240 min.

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Extracellular 120 % of initial concentration (by UV analysis) 100 80 60 40 20 0 0 50 100 150 200 250 Intracellular 120 % of final fluoresence intensity (From FACS) 100 80 60 40 20 0 300

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

(a)
120 % of initial concentration (UV analysis) 100 80 60 40 20 0 0 50 100 Extracellular Intracellular

Time in minutes

120 100 80 60 40 20 0 300 % of final fluoresence intensity (from FACS)

150

200

250

(b)

Time in minutes

Fig. 4. Decrease in FITC concentration in the cell supernatant (from UV/visible spectroscopy shown with open squares on the left hand yaxis) and increase in the FITC intensity inside the cell (calculated from mean uorescence intensity using ow cytometry and is shown with closed squares on the right hand y-axis) for (a) IbuprofenFITC and (b) IbuprofendendrimerFITC conjugate.

drug or drugdendrimer conjugate) in a ow cytometer. Both the techniques showed a reasonably good correlation 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 by measuring 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 60 min. From the mean uorescence intensity, it was observed that there was no further increase after 2 h (Fig. 4b). Although, both free and dendrimer-conjugated ibuprofen shows similar proles, 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 dendrimer molecule. The cellular entry was also visualized by using confocal microscopy. (Fig. 5ad) It is evident that the FITC-labeled ibuprofen, dendrimer, and the dendrimeribuprofen conjugates entered the cells and localizing mostly in the cytoplasm, while the nucleus appears to be relatively free of the presence of any uorescence 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

Fig. 5. Confocal uorescence 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 uorescence 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, uid phase 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 dendrimeribuprofen conjugate [28]. As there are no specic ligands in this dendritic nanodevice, it is not expected to be transported by receptor-mediated endocytosis. Epithelial 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 uid phase endocytosis by non-specic interactions similar to hyperbranched polymeribuprofen conjugates described by Kolhe et al [30]. The results are further substantiated by our recent ndings that the cellular uptake of FITClabeled PAMAM G4-OH dendrimer was reduced in presence of uid phase endocytosis inhibitor (unpublished data). Once inside the cell, the drugdendrimer conjugate is expected to be transported by the endosomes 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 the lysosomal 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-inammatory activity of ibuprofen dendrimer conjugate Ibuprofen is a non-steroidal anti-inammatory 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 inammation, so reducing prostaglandin synthesis reduces inammation and the perceived pain associated with the inamed 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 efciency of the dendrimeribu-

120 Percent inhibition of PGE 2 100 80 60 40 20 0 30

Ibuprofen G4-Ibuprofen conjugate

60 Time in minutes

360

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 360 min (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.

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 dendrimeribuprofen conjugate showed a signicant 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 of free ibuprofen enters the cells within 30 min, it may not achieve a sufcient concentration to elicit a rapid pharmacological response. On the other hand, dendrimeribuprofen conjugate, achieves a high local concentration 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 suppress prostaglandin 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 efcacy than free drug. The authors concluded that the dendrimerMTX conjugate enters the cells efciently and the ester bond is hydrolyzed at the low pH found in the endosome, thereby releasing free MTX [24]. Assuming comparable hydrolysis of ester

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bond inside the cells, our high payload drug conjugates would show a signicantly 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 concentration at the target site and potentially overcome the systemic adverse effects of free ibuprofen. For parenteral administration, 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 signicantly. This will decrease the dosage required to achieve the same effect and more efcient delivery of the drug will lead to a greater therapeutic efcacy 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 PGE2 suppression, suggesting higher activity for the conjugated 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 efcacy. Future studies are warranted to evaluate the pharmacokinetic and pharmacodynamic aspects of these nanomaterials 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 supplemental funding (NIH 3U01HD-37261-04SI). We would like to thank Prof. David Bassett for the help with FACS measurements.

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