Targeting and Inhibition of Cell Growth by an Engineered Dendritic

Patri, A. K.; Majoros, I. J.; Baker, J. R., Jr. Dendritic polymer macromolecular ...... V. Andrés-Guerrero , D. Barbosa-Alfaro , I.T. Molina-Martíne...
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J. Med. Chem. 2005, 48, 3729-3735

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Targeting and Inhibition of Cell Growth by an Engineered Dendritic Nanodevice Thommey P. Thomas,* Istvan J. Majoros, Alina Kotlyar, Jolanta F. Kukowska-Latallo, Anna Bielinska, Andrzej Myc, and James R. Baker, Jr. Center for Biologic Nanotechnology, University of Michigan Medical School, 9220 MSRB III, Ann Arbor, Michigan 48109-0648 Received October 18, 2004

The cellular uptake and cytotoxicity of an engineered multifunctional dendritic nanodevice containing folic acid (FA) as the targeting molecule, methotrexate (MTX) as the chemotherapeutic drug, and fluorescein (FI) as the detecting agent were studied in vitro. FI and FA were conjugated to the generation 5 poly(amidoamine) (G5) dendrimer carrier through a thiourea and amide linkage and MTX was conjugated through an ester linkage to the carrier to generate the trifunctional dendritic device, G5-FI-FA-MTX. This trifunctional dendrimer-drug conjugate bound to FA receptor-expressing KB cells in a dose-dependent and saturable manner. Confocal microscopic analysis demonstrated cellular internalization of the conjugate. G5-FIFA-MTX induced a time- and dose-dependent inhibition of cell growth in KB cells. The targeted dendrimer conjugates G5-FI-FA-MTX and G5-FA-MTX inhibited cell growth in KB cells, whereas the nontargeted G5-MTX failed to induce growth inhibition. These studies show the potential of G5-FI-FA-MTX or G5-FA-MTX for targeting and growth suppression of tumor cells that overexpress FA-receptors. Introduction Current chemotherapeutic approaches for cancer treatment lack efficacy due in part to the drugs’ nonspecific cytotoxicity. Recent advances in the development of biocompatible polymers, combined with the identification of cancer-specific molecular targets, have allowed the application of polymers as agents for the specific targeting of cancer cells.1-5 In this approach, a polymer is modified to incorporate a targeting molecule and a therapeutic drug, the former to guide the polymer to the tumor site and the latter to induce cell death. The homing of the polymer-therapeutic conjugates specifically to cancer cells reduces the toxicity and increases the therapeutic index. Polymeric drug conjugates have several advantages over free drugs, such as increased solubility in aqueous media, increased plasma half-life, and decreased drug resistance. Several synthetic and natural polymers have been tested in the past decade for targeting tumor cells.5-7 An optimal targeted drug delivery system requires a platform that is able to carry multiple components, such as a drug, a cancer-detecting agent, and a fluorescent sensing agent. Poly(amidoamine) (PAMAM) dendrimers offer such a carrier system, having a defined branched chain structure capable of carrying multiple molecular entities that are either linked covalently to its surface or are encapsulated in its interior space. The dendrimers are suitable as carriers for several biomedical applications.5,6-11 Dendrimer conjugates have been used for delivering drugs,11,12 DNA,3,13,14 radionuclides,15 MRI contrast agents,16 and boron for neutron capture therapy.17 The vitamin folic acid (FA) is a molecule that has been extensively investigated for targeting cancer cells. The * Corresponding author. Tel: 734-615-3594. Fax: 734-936-2990. E-mail: [email protected]. Website: www.nano.med.umich.edu.

high affinity receptor for FA (FAR, Kd ) 0.1-1 nM) is known to be overexpressed in several human carcinomas, even up to a 100-fold.18-21 FAR is expressed on the basolateral surface (blood side) of transformed cells as compared to the apical surface expression in normal cells.22 This property complements the cancer cell specificity of FA as a targeting agent when delivered through the blood. FA is easily available and inexpensive, and its small molecular size allows easy tumor penetration and favorable pharmacokinetics. For these reasons, FA has been widely used for the targeting of several bioactive agents, such as protein toxins, oligonucleotides, plasmids, liposome-entrapped drugs, radiopharmaceutical agents, and MRI agents.16, 23-29 The FA analogue methotrexate (MTX) is a widely used chemotherapeutic drug for the treatment of a variety of malignancies.30 Methotrexate inhibits the cytosolic enzyme dihydrofolate reductase (DHFR), resulting in the depletion of the reduced folic acid that is required for nucleotide synthesis through one-carbon transfer, thus leading to the inhibition of DNA replication and subsequent cell death.31 In this study we show the in vitro biological properties of a dendrimer conjugate in which FA and MTX are used as the targeting and therapeutic agents, respectively. The device is effective in the specific targeting and killing of tumor cells that overexpress the FA receptor. Materials and Methods Abbreviations. FI, fluorescein isothiocyanate; FA, folic acid; G5, generation 5 poly(amidoamine) dendrimer (G5); MTX, methotrexate; G5-FI-FA, bifunctional dendrimer containing FI and FA; G5-FI-FA-MTX, trifunctional dendrimer with MTX linked through an ester linkage; EDTA, ethylenediaminetetraacetic acid; XTT, sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate.

10.1021/jm040187v CCC: $30.25 © 2005 American Chemical Society Published on Web 04/23/2005

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Figure 1. Schematic representation of generation 5 (G5) dendrimer surface-functionalized with FITC, FA, and MTX. Materials. The KB cells were obtained from ATCC (CLL17; Rockville, MD). Trypsin-EDTA, Dulbecco’s phosphate-buffered saline (PBS), fetal bovine serum, cell culture antibiotics, and RPMI medium were obtained from Gibco/BRL (Gaithersburg, MD). All other reagents were from Sigma (St. Louis, MO). The synthesis and characterization of the dendrimer conjugates is reported as a separate communication.32 A schematic representation of the structure of the trifunctional conjugate G5-FI-FA-MTX is shown in Figure 1. All the dendrimer preparations used in this study were synthesized at our center and have been surface-neutralized by acetylation of the free surface amino groups. Cell Culture and Treatment. KB cells were maintained in folate-free medium containing 10% serum as described before33 to provide extracellular FA similar to that found in human serum.34 Cells were plated in 12-well plates for uptake studies, in 24-well plates for cell growth analysis, and in 96-well plates for 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay. Cells were rinsed with FA-free medium containing dialyzed serum and incubated at 37 °C with dendrimer-drug conjugates for the indicated time periods and concentrations. KB cells were also maintained in RPMI medium containing 2.5 µM FA to obtain cells that express low folic acid receptor (FAR). Flow Cytometry and Confocal Microscopy. The standard fluorescence of the dendrimer solutions was quantified using a Beckman spectrofluorimeter. For flow cytometric analysis of the uptake of the targeted polymer, cells were trypsinized and suspended in PBS containing 0.1% bovine serum albumin (PBSB) and analyzed using a Becton Dickinson FACScan analyzer. The FL1-fluorescence of 10 000 cells was measured, and the mean fluorescence of gated viable cells was quantified. Confocal microscopic analysis was performed in cells plated on a glass cover-slip using a Carl Ziess confocal microscope. Fluorescence and differential interference contrast (DIC) images were collected simultaneously using an argon laser and appropriate filters for FITC. Evaluation of Dendrimer Cytotoxicity. Cell growth was determined by assay of the total protein in lysates of treated cells using a bicinchoninic acid reagent (PIERCE, Rockford, IL) and by XTT assay, using a kit from Roche Diagnostics (Indianopolis, IN).

Results Cellular Uptake of the Dendrimer-Drug Conjugates. The fluorescence of the standard solutions of

Figure 2. Dose-dependent binding of G5-FI-FA-MTX in KB cells. The cells were maintained in FA-free medium and incubated with different concentrations of the indicated dendrimers for 1 h. The cells were then rinsed and resuspended in PBSB, and the fluorescence was measured in a flow cytometer (panel A). In panel B, the mean cell fluorescence, after being normalized for the fluorescence of standard solutions of the dendrimers, is presented.

the conjugates G5-FI, G5-FI-FA, and G5-FI-FAMTX were measured using a spectrofluorimeter. A linear relationship between the dendrimer concentration and the fluorescence was observed at 10-1000 nM. The fluorescence of 100 nM solutions of G5-FI, G5-FI-FA, and G5-FI-FA-MTX were respectively 0.57, 0.23, and 0.11 spectrofluorimetric units. These differences in fluorescence may be indicative of either quenching due to the presence of FA and MTX on the dendrimer or due to photobleaching during the additional synthetic reactions following FI conjugation, but not due to any loss of FI.32 The cellular uptake of the dendrimers was measured in KB cells that express a high cell surface FAR. The FA-conjugated dendrimers bound to the cells in a dosedependent fashion with 50% binding at 10-15 nM for both the G5-FI-FA and G5-FI-FA-MTX, while the control dendrimer G5-FI was not detected in the KB cells (Figure 2A). Identical binding curves were obtained for the G5-FI-FA and G5-FI-FA-MTX when the fluorescence obtained was normalized for the quenching observed in the standard solutions of the dendrimers (Figure 2B). Analysis of the kinetics of the binding of the G5-FI-FA-MTX (100 nM) showed that maximal binding was achieved within 30 min (data not shown), which is similar to reports for the binding of another FA-linked nanoparticle in KB cells.35 To confirm the receptor specificity for the conjugate, the effect of free FA on the uptake of the dendrimers was tested in KB cells that express both high and low

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Figure 3. Effect of free FA on the uptake of G5-FI-FA and G5-FI-FA-MTX in KB cells expressing high and low FAR. KB cells that express high (solid bars) and low (shaded bars) FAR were incubated with 30 nM of the dendrimers for 1 h at 37 °C and rinsed, and the fluorescence of cells was determined by flow cytometric analysis (left panel). Preincubation with 50 µM free FA for 30 min totally prevents cellular binding and uptake of the polymer conjugates (right panel).

FAR. The binding of the conjugates to the low-FARexpressing KB cells was 30% of that of the high-FARexpressing cells for both the G5-FI-FA and G5-FIFA-MTX (Figure 3, left panel). We found that 50 µM FA completely blocked the uptake of either targeted dendrimers (30 nM) in both the low- and high-FARexpressing cells (Figure 3, right panel). The binding and internalization of the conjugates to KB cells was also assessed by confocal microscopy. As shown in Figure 4, conjugates containing the targeting molecule FA internalized into the cells within 24 h. As compared to the cells treated with the control conjugates, the cells exposed to G5-FI-FA-MTX were less adherent and rounded up, indicating cytotoxicity induced by the drug-conjugate. Inhibition of Cell Growth by Drug-Conjugated Dendrimers. Because the binding of the conjugate to KB cells reaches maximal uptake within 1 h,33 the effect of the G5-FI-FA-MTX on cell growth was initially tested by preincubation of cells with the conjugate for 1 h, followed by incubation in a drug-free medium for 5 d. Under such conditions, the conjugate failed to show any growth-inhibitory effect in KB cells. When the cells were preincubated with dendrimers for 4 h, there was a modest decrease of about 10% in cell growth, as determined by XTT assay (data not shown). The cytotoxicity measurements were, therefore, performed by incubation with the dendrimer for a minimum of 24 h, a preincubation time period shown to induce significant cytotoxicity. Previous studies have shown that MTX-induced cytotoxicity is detectable in vitro only if the medium is completely deprived of FA.36 The effect of the trifunctional dendrimers on cell growth was tested in cells incubated in a FA-deficient medium. Under these conditions, the G5-FI-FA-MTX and free MTX inhibited cell growth in a time- and dose-dependent fashion, whereas the control dendrimers failed to inhibit the cell growth (Figure 5). The inhibition of cell growth induced by the conjugates was also tested by XTT assay, which is based on the conversion of XTT to formazan by the active mitochondria of live cells.37 As shown in Figure 6, the

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G5-FI or G5-FI-FA were not growth inhibitory for the cells at 1, 2, or 3 days, whereas the G5-FI-FA-MTX and free MTX showed time-dependent cytotoxicity. As the growth inhibition induced by free MTX was higher than with the equimolar concentrations of MTX in the G5-FI-FA-MTX below 1 µM (Figure 5), we tested the hypothesis that, under the FA-deprived conditions of the assay, the FA moiety in the G5-FIFA-MTX may be rescuing the cells from MTX-induced cytotoxicity. As the G5-FI-FA-MTX preparation contained equimolar concentrations of MTX and FA, the effect of similar concentrations of free MTX and free FA on the inhibition of cell growth was determined. As shown in Figure 7, at equimolar concentrations of free FA and MTX, the FA reversed the inhibition of cell growth induced by MTX. The presence of 150 nM FA almost completely reversed the growth arrest caused by 150 nM MTX. Moreover, the cytotoxicity induced by G5-FI-FA-MTX (filled square symbols) and equimolar combinations of FA and MTX (filled circle symbols) was similar. As free FA blocks the uptake of the dendrimers as well as rescues cells from MTX-induced cytotoxicity, the effect of preincubation of cells with excess FA on the anti-proliferative effect of G5-FI-FA-MTX was tested. As shown in Figure 8, excess free FA not only blocked the growth inhibition by G5-FI-FA-MTX but also increased cell growth 20% above that of the control cells (Figure 8). To check if free MTX was released from the dendrimer prior to its entry into the cells, the stability of the dendrimer was tested in the cell culture medium. For this analysis, the G5-FI-FA-MTX was incubated with cell culture medium for 1, 2, 4, and 24 h, and the incubation medium was filtered using a 10 000 MW cutoff ultrafiltration device. The effect of the retentate and the filtrate on the growth of the KB cells was tested. During the 24-h incubation time periods, the retentate was cytotoxic, whereas the filtrate failed to show any cytotoxicity (Figure 9), indicating the lack of release of the free MTX from the conjugates. There was a slow release of the MTX after 24 h, reaching a maximum of 40-50% release in 1 week (data not shown). The antiproliferative effect of the MTX conjugates was compared to conjugates that lacked either the FA or the FI molecule. As shown in Figure 10, the MTX-conjugated dendrimer that lacked FA failed to induce cytotoxicity, whereas the targeted dendrimer in the absence or presence of the dye molecule FI induced cytotoxicity. The failure of G5-MTX to inhibit cell growth further supports the inference of the previous study (Figure 9) that any extracellular release of MTX from the conjugate is not contributing to the inhibition of cell growth by the FA-targeted conjugates. Discussion The observations that G5-FI-FA-MTX bound to FA-expressing cells in a time- and dose-dependent fashion, that the control dendrimer G5-FI failed to bind to KB cells, that the dendrimer failed to associate with KB cells that do not overexpress FAR, and that free FA competed with the conjugate for binding (Figures 2 and 3) all indicate specific, receptor-mediated binding of the conjugate. The concentration-dependent and saturable

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Figure 4. Confocal microscopy of KB cells treated with dendrimer conjugates. KB cells were incubated with 250 nM of the indicated dendrimers for 24 h and confocal images were taken. The left and right panels under each treatment represent FITC fluorescence and a DIC image of the same observation field.

Figure 6. Growth inhibition of KB cells by drug-conjugated dendrimers determined by XTT assay. Cells were exposed to 1 µM each of the conjugates or 5 µM of free MTX 1 (open bars) for 2 days (shaded bars) and 3 days (filled bars), and the XTT assay was performed. The data are expressed as percent absorbance of control cells for each time point that did not undergo any treatment. Other conditions are as given in the legend of Figure 4. The values shown are the mean of duplicate cell samples. Similar results were obtained in an independent experiment.

Figure 5. Antiproliferative effect of MTX-conjugated dendrimers. (A) Time course of inhibition of cell growth. Cells were treated with 300 nM conjugates (equivalent of 1500 nM MTX) or 1500 nM free MTX for 1-4 days, and cell proliferation was determined by estimation of cellular protein content. (B) Dosedependent inhibition of cell growth by MTX (circle symbols), G5-FI-FA-MTX (square symbols), and G5-FI-FA (diamond symbols). Cells were treated for 2 days with different concentrations of the conjugates or free MTX. The concentrations of the two conjugates are given as actual concentration (top axis), and as MTX equivalents of the G5-FI-FA-MTX (filled square symbols), with 5 MTX per dendrimer molecule. The open square symbols show the G5-FI-FA-MTX toxicity corrected for that shown by G5-FI-FA. The data represents mean of duplicates from a single experiment. Similar data were obtained in a separate experiment.

binding of the conjugate is similar to previous results obtained for the binding of free FA in KB cells.38,39 G5-FI-FA and G5-FI-FA-MTX bound to the KB

cells with similar binding curves (Figure 2B), indicating that the addition of MTX did not alter the affinity of the conjugate. Confocal microscopic analysis showed cellular internalization of the dendrimers, demonstrating the utility of the nanodevice as an intracellular drug delivery agent. In KB cells, FA and MTX can be internalized through both the FA receptor (FAR) and the reduced folate carrier (RFC), having varying affinities for the two ligands.40-43 FA is poorly transported through RFC, and the Kt value for MTX transport through the RFC is 1-5 µM,44 suggesting that, below 1 µM, the RFC is not participating in the uptake of G5-FI-FA-MTX. This is supported by the similar affinities observed between G5-FI-FA and G5-FI-FA-MTX (Figure 2B) and is consistent with the observation that MTX conjugated with a molecule such as FITC can impede its transport through the RFC.45

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Figure 7. Comparison of the inhibition of cell growth by G5-FI-FA-MTX and equimolar concentrations of mixtures of MTX and free FA. KB cells were treated with 150 or 500 nM MTX in the presence (filled circle symbols) or absence (open circle symbols) of equimolar concentrations of free FA for 24 h. Cells were also treated with 30 and 100 nM G5-FIFA-MTX (equivalent to 150 and 500 nM MTX) in parallel. The cells were rinsed to remove the drugs and incubated with fresh medium for an additional 6 d, and total cell protein was determined.

Figure 9. Stability of drug-conjugated dendrimers in cell culture medium. G5-FI-FA-MTX was incubated with medium at 2 µM concentration for 24 h. The incubation medium was filtered through a Centricon 10K MW cutoff filter. The retentate (adjusted to prefiltration volume) and the filtrate were incubated with KB cells (at 200 nM conjugate, as determined from the concentration of the prefiltration sample) for 2 days, and the XTT assay was performed. Similar results were obtained for the retentate and filtrate obtained from the medium that had been preincubated with the dendrimers for 1, 2, and 4 h. The values shown are the mean of duplicate cell samples, with similar results obtained in an independent experiment.

Figure 8. Reversal of G5-FA-MTX-induced inhibition of cell growth by free FA. KB cells were exposed to different concentrations of the conjugate or free MTX for 24 h in the absence (filled symbols) or presence (open symbols) of 50 µM FA. The incubation medium was removed, the cells were rinsed and incubated with fresh medium for an additional 5 days in the absence of the drugs, and the XTT assay was performed. Key: 0, 9, cells treated with MTX; 4, 2, cells treated with G5-FI-FA-MTX. The values show a mean of four replicate cell samples. *p < 0.05 vs respective controls in the absence of FA.

FAR is a single-chain membrane glycoprotein containing a single high-affinity binding site for FA, with Kt values for FA and MTX, respectively, of 1 and 300 nM.38,46,47 As the low-FAR-expressing KB cells bound both G5-FI-FA and G5-FI-FA-MTX to the same extent (30%) vs FAR-positive cells (Figure 2), the MTX moiety of the conjugate is unlikely to be participating in binding to the FAR. This is supported by the observation that the control dendrimer G5-MTX failed to induce any antiproliferative effect (Figure 10). Nonetheless, the conjugate binding may involve more complex processes because of the multiple receptors that can be occupied by either multiple FA or MTX moieties present in the dendrimer. Although the conjugate binding to the cells reaches a maximum in 1 h, preincubation with the conjugate for up to 4 h, followed by 5 d in conjugate-free medium, failed to show any significant antiproliferative effect. It is possible that threshold MTX levels required for inhibition of cell growth are not achieved in such a short period. The cytotoxicity of the MTX depends on the duration that a threshold intracellular level is main-

Figure 10. Comparison of induction of cytotoxicity by conjugates with and without the targeting agent FA or the detecting agent FI. KB cells were incubated with 30 nM of the conjugates ()150 nM effective MTX concentration) for 24 h, and the incubation medium was removed. The cells were rinsed and incubated for an additional 5 days in fresh medium in the absence of the drugs, and the XTT assay was performed. The values show a mean ( SE of four replicate cell samples. *p < 0.05 vs cells treated with PBS, G5-FI, and G5-MTX.

tained.31,48,49 Cells contain high concentrations of DHFR, and to shut off the DHFR activity completely, anti-folate levels 6 orders of magnitude higher than the Ki for DHFR are required.43 Furthermore, less than 5% of the enzyme activity is sufficient for full cellular enzymatic function.50 Under long-term preincubation, sustained intracellular levels of the drug conjugate can be achieved through recycling the FAR.51 The reduced antiproliferative effect of G5-FI-FAMTX as compared to free MTX may be due to the slow hydrolysis and release of the FA from G5-FI-FA-MTX and the FA acting as a rescuing agent to reverse the cytostatic effect of the anti-folate MTX.37 This is supported by the observation that equimolar concentrations of free FA and free MTX evoked growth-inhibitory effect similar to that of G5-FI-FA-MTX (Figure 7). The potency of free MTX to induce cytotoxicity relies on its polyglutamation, which results in the entrapment and accumulation of the drug in the cell.30 The physiological polyglutamation of MTX takes place at its γ-carboxyl group. As the chemical conjugation of MTX

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to the dendrimer is through the γ-carboxyl group of the former, such polyglutamation of the MTX moiety in the conjugate is not possible. Nonetheless, the conjugation of the MTX to the dendrimer may by itself impede its exit from the cell through the exit pump. This is supported by confocal microscopic data, which showed the retention of FA-conjugated dendrimers in KB cells preincubated with the conjugate for 24 h, followed by incubation in a dendrimer-free media for 4 days (unpublished observation). One of the limiting factors in the application of an anti-folate such as MTX is the development of drug resistance. Our results suggest that the MTX in the dendrimer is primarily carried into the cell through FAR-mediated endocytosis with minimal participation of the RFC. FAR is known to not be involved in MTX-induced drug resistance.52 Therefore, the drug resistance due to the altered expression of RFC53 may be overcome in targeted therapy using the conjugate. Similarly, as the conjugate retention in the cell is independent of polyglutamation (see above), the resistance caused by reduced polyglutamation, similar to that which has been observed in multiple human leukemia cells,54 can be avoided. These results demonstrate the applicability of dendrimer as a suitable polymer for the specific delivery of molecules into cells. The development of a multifunctional dendrimer conjugate is promising for combining cancer imaging and targeted drug delivery. This type of targeted delivery may overcome the nonspecific cytotoxicity and some forms of drug resistance caused by the free drug. The application of the targeted delivery may also be extended to other therapeutic applications. For example, as MTX is currently used also as an antiinflammatory agent, development of a lymphocytespecific dendrimer-MTX targeting device may overcome any nonspecific effect of the free drug.

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Conclusion The trifunctional dendrimer-drug conjugate G5-FIFA-MTX binds, internalizes, and induces cytotoxicity in FA receptor-expressing KB cells in vitro in a receptor specific manner. The development of a multifunctional dendrimer conjugate is promising for combining cancer imaging and targeted drug delivery. This type of targeted delivery may overcome the nonspecific cytotoxicity and some forms of drug resistance caused by the free drug. Acknowledgment. This work was supported in part by the National Cancer Institute Contract N01-CM97065-32. The authors gratefully acknowledge the technical assistance of James Beals. References (1) Moghimi, S. M.; Hunter, A. C.; Murray, J. C. Long-circulating and target-specific nanoparticles: Theory to practice. Pharm. Rev. 2001, 53, 283-318. (2) Brigger, I.; Dubernet, C.; Couvreur, P. Nanoparticles in cancer therapy and diagnosis. Adv. Drug Delivery Rev. 2002, 54, 631651. (3) Luo, Y.; Prestwich, G. D. Cancer-targeted polymeric drugs. Curr. Cancer Drug Targets 2002, 2, 209-226. (4) Jensen, K. D.; Nori, A.; Tijerina, M.; Kopeckova, P.; Kopecek, J. Cytoplasmic delivery and nuclear targeting of synthetic macromolecules. J. Controlled Release 2003, 87, 89-105. (5) Majoros, I. J.; Thomas, T. P.; Baker, J. R., Jr. Molecular engineering in Nanotechnology: Engineered Drug Delivery. In Handbook of Theoritical and Computational Nanotechnology; Rieth, M., Schommers, W., Eds.; in press.

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