Dendritic polymer macromolecular carriers for drug
Transcription
Dendritic polymer macromolecular carriers for drug
466 Dendritic polymer macromolecular carriers for drug delivery Anil K Patri, István J Majoros and James R Baker Jr* Dendrimers are synthetic, highly branched, mono-disperse macromolecules of nanometer dimensions. Started in the mid-1980s, the research investigations into the synthetic methodology, physical and chemical properties of these macromolecules are increasing exponentially with growing interest in this field. Potential applications for dendrimers are now forthcoming. Properties associated with these dendrimers such as uniform size, water solubility, modifiable surface functionality and available internal cavities make them attractive for biological and drug-delivery applications. Addresses Center for Biologic Nanotechnology, Department of Internal Medicine, 9220C MSRB III, University of Michigan, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109-0648, USA www.nano.med.umich.edu *e-mail: [email protected] Current Opinion in Chemical Biology 2002, 6:466–471 1367-5931/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S1367-5931(02)00347-2 Abbreviations EDC 1-[3(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride FITC fluorescein isothiocyanate hFR high-affinity folate receptor PAMAM polyamidoamine PEG polyethylene glycol PSMA prostate-specific membrane antigen Introduction A macromolecular drug-delivery system is a complex material in which a drug is attached to a carrier molecule such as a synthetic polymer, antibody, hormone or liposome. As the absorption and distribution of the drug in such a system depend on the properties of the macromolecular carrier, parameters such as site specificity, protection from degradation and minimization of side effects can be altered by modifying the properties of the carrier. An ideal drug carrier must be biochemically inert and non-toxic, while protecting the drug until it reaches the desired site of action, with the carrier then releasing the drug. Many polymeric drug-delivery systems have been developed over the years and have been extensively reviewed [1]. By contrast, the use of dendrimers [2••,3••], which possess many of the above-mentioned properties for an ideal drug-carrier system, has not been highlighted. We review the current trends in the drug-delivery systems using dendrimers. Dendrimers in drug delivery Dendrimers are synthetic, highly branched, spherical, mono-disperse macromolecules of nanometer dimensions, prepared by the iterative synthetic methodology. Since the pioneering work of Tomalia et al. [4] and Newkome et al. [5] on dendrimer synthesis in the early 1980s, several research groups have contributed both synthetic methodology and specific applications for this field. There is a continuing effort to improve the efficiency and lessen the cost of synthesizing these macromolecules. Further investigations also are examining the specific physical and chemical properties of dendrimers and, although there are many factors that remain unknown, potential applications for dendrimers are now forthcoming. These macromolecules have uniform size and are mono-dispersed. They also have modifiable surface functionality as well as internal cavities [6]. These characteristics, along with water solubility, are some of the features that make them attractive for biological and drug-delivery applications [7,8]. In fact, much of the current work in this field is focused on the characteristics of dendrimer-based devices in vivo. Polymeric micelles provide a model of sorts for dendrimermediated drug delivery. In polymeric micelles, the drug molecules can be trapped in the inner hydrophobic core while the outer shell is hydrophilic and soluble in aqueous media. However, below a critical micelle concentration, the polymeric aggregates dissociate into free chains, leading to the sudden release of the drug. Newkome et al. [9] have synthesized a dendritic unimolecular micelle (Figure 1a) containing hydrophobic interior and hydrophilic surface functionality to overcome this problem. Unlike polymeric micelles, these unimolecular micelles do not dissociate as they are covalently bound. The internal hydrophobic cavities of this unimolecular micelle were shown to solubilize various hydrophobic guest molecules. Newkome’s group has also prepared a series of dendritic molecules possessing internal heterocyclic loci [10] capable of specific binding of guest molecules (Figure 1b) and dendrimers with terminal tryptophan units [11]. This provides a basis for dendrimers as drug carriers. Specific types of dendrimers for drug delivery Perhaps the family of dendrimers most investigated for drug delivery is the polyamidoamine (PAMAM) dendrimer (Figure 1c). The divergent synthesis of PAMAM dendrimers starts with an amine functional core unit that is reacted with methyl acrylate by Michael addition reaction. This results in the formation of two new branches per amine group with ester-terminated dendrimer, which is called ‘half-generation’ dendrimer. Subsequent amidation of the methyl ester with ethylene diamine gives a ‘fullgeneration’ amine-terminated dendrimer. Repetition of Michael addition and amidation steps gives the next-higher ‘generation’ dendrimer with increase in the molecular weight, number of terminal functional groups and size. PAMAM dendrimers are biocompatable, nonimmunogenic, water-soluble and possess terminal- modifiable amine functional groups for binding various targeting or guest molecules. The internal cavities of PAMAM Dendritic polymer macromolecular carriers for drug delivery Patri, Majoros and Baker 467 Figure 1 R +NR -O C 4 2 +NR -O C 4 2 + NR4 O2C R O + +NR -O C 4 2 O O CO2-R4N+ R O NH R' NR4-O2C R + N H R O H O H N N CO2 R4N R' H O O O O O O O H O H O O + + CO2-R4N+ NR4-O2C NR4-O2C + H R' O R O O CO2-R4N+ CO2-R4N+ CO2-R4N+ NR4-O2C + NR4-O2C HN O + NR4-O2C + NR4-O2C +NR -O C 4 2 O O HN CO2-R4N+ O O O R H2N H2N H2N H2N H2N H2N H2N H2N NH NH O H2N NHO NH O NH O H2N H2N H2N H N H N O H2N H2N H2N H2N H2N H2N H2N O N H HN O HN HN O O H2N H2N HN N N O N H N O HN O HN O HN N N O O NH O O O HN N NH N O HN O N O O N H HN O N N H N N O HN O N H O O H2N H2N N HN N H HN O HN O N N H2N NH N O H2N N O O O O HN H N O O O N H2N O H N O O HN O O HN N O HN HN O N N N H H N H2N N O O N NH HN N O O O NH HN NH N HN N O O N O O N NH O O O NH HN HN HN O N O HN HN O H2N N N O H2N HN N HN O NH2 NH2 O O HN O O HN O O HN NH2 HN NH2 HN HN HN HN H2N H2N N O HN HN NH2 NH 2 O O NH HN N NH2 NH 2 NH2 NH2 N O O O O HN HN HN NH2 NH2 NH2 NH2 O N O NH O NH NH O O NH HN N O N O O O NH H2N N N N NH2 NH2 N N N H2N NH2 N N H2N H2N NH2 N N N N H2N H2N N N N N H2N NH2 NH2 N N N N H2N H2N NH2 NH2 N N N N N H2N N N H2N NH2 N N N N NH2 NH2 N N N N H2N NH2 N N N H2N NH2 N N NH2 N N H2N NH2 N N H2N H2N NH2 N N N H2N N N N NH2 NH2 N N H2N N N H2N NH2 N N NH2 NH2 NH2 H2N H2N H2N (c) NH2 N N 2 NH2 N H2N NH2 NH2 NH NH2 NH NH2 O NH O NH2 H NH N N O NH2 N N O NH N O O O NH NH NH2 H N NH HN NH2 N N O NH O NH O O O HN O NH NH2 N N O NH2 O N O NH O NH O NH NH2 NH H N O NH N N NH2 O O NH H O O NH N N NH O N H N H N O N O N N NH2 N H O O NH H NH2 N O OH N O N N O N H N O NH2 O N H O NH NH2 N O H H N HN N O N O N NH2 O H O N O N N N H H H NH2 N N O N H N H O O N N H N O NH2 O N N NH HH H N O NH2 O N O N O H N HN N O N O N NH2 H O NH N O NH2 O HN H O HN N NH NH O N O N O NH2 N O H N N O NH2 N H N N HN H O O HN NH NH O O O N NH2 O HN O NH2 O HN HN O HN NH N N NH OHN NH2 N N O O NH2 HN O N O N O HN HN O NH2 NH HN O O NH2 HN O O NH2 O NHHN N HN NH HN NH2 N OHN N NH2 O O N HN NH2 N O O N HN O HN NH2 O HN HN O O O HN NH2 O O HN NH2 HN HN HN NH2 NH2 HN HN NH2 NH2 NH2 NH 2 NH2 NH2 NH O N N H H N O N H N NH HN O HN O N HN HN O N NH HN O O N O NH HN O N NH O N O O N O NH2 N N H2N NH NH NH2 O NH2 O NH NH O N O NH NH2 O NH NH2 N N N NH HN O N N H N H2N O O O O NH2 N N NH2 NH2 O N N N O O O N O HN H2N NH NH NH NH H2N NH NH O NH O H2N O O NH NH O O O O NH O O NH HN NHHN R NH2 NH2 H2N H2N H N 2 H 2N H N 2 N N N H2N H2N NH N NH O N HN O H N N HN N O N HH N N HN H2N O H2N H2N H2N H2N NH O N N ON HN NH O NH NH O H N H N O H2N H N HO N O O HN N O HN N O H N H N O H2N O O O H O N H N O H2N H2N HN NH N O H N H N O H2N H2N O NH O NHO N NH N O NH O NH HN NH O N O O N HN N N O NH N NH O NH O O NH O NH2 H2N H2N H2N H2N H2N NH NH NH NH NH O O NH H2N H2N H2N H2N H2N R (b) H2N H2N H2N R O O O R R R R O R (a) O O NH O O O O R N H HN O O R R H2N H2N HN O R R O R' O HN CO2-R4N+ CO2-R4N+ O O N H R R O O O O O N H H R + H + CO2 R4N +NR -O C 4 2 N O N - NR4 O2C H O N H + N N H CO2-R4N - R N O H + - O O N N + R O NH O H H CO2-R4N NR4-O2C O O O O O + NH O O O CO2-R4N+ O H N O R +NR -O C 4 2 NH O O H N R R O O NH O R O HN R CO2-R4N+ CO2-R4N+ NR4-O2C + NR4-O2C R O O O O R CO2-R4N+ - R R R O O R CO2-R4N+ CO2-R4N+ H2N NH2 H2N NH2 NH2 (d) Current Opinion in Chemical Biology Different families of dendrimers. (a) Unimolecular micelle. (b) Newkome’s dendrimer with internal binding units. (c) PAMAM dendrimer. (d) POPAM dendrimer. dendrimers can host metals or guest molecules because of the unique functional architecture, which contains tertiary amines and amide linkages. Twyman et al. [12] have converted the ester-terminated half-generation PAMAM dendrimers to a more water-soluble hydroxyl surface by reacting with tris. They have investigated complexing these novel dendrimers with small hydrophobic guest molecules, which then become highly soluble in phosphatebuffered dendrimer solution at pH 7.0. The complexes are not stable in acid and precipitate at pH 2, presumably 468 Next-generation therapeutics Figure 2 (a) (b) (c) (FA) Imaging (FA) (CO2H) G5 Gx O Targeting device Gx O-C-Drug O (CO2H) G5 Imaging O-C- Drug (FA) (FA) G5 G5 G5 (CO2H) G5 (CO2H) G5 (CO2H) (FA) (CO2H) (FA) Current Opinion in Chemical Biology Dendritic nanodevices for therapeutic applications. (a) Surface-modified PAMAM dendrimer for targeting, imaging and drug delivery. (b) Antibody–dendrimer conjugate. (c) Tecto(dendrimer). FA, folate. because of the protonation of the internal tertiary amines, which releases the guest molecules. Gadolinium is an FDA-approved contrast agent for MRI. It provides greater contrast between normal tissue and abnormal tissue in the brain and body. It is safer than the iodine-type contrast used in CT scans, is non-radioactive and is rapidly cleared by kidneys. An early attempt to use dendrimers in vivo was efforts in the development of target-specific MRI contrast agents by Wiener et al. [13]. These investigators produced gadolinium complexes of folate-conjugated PAMAM dendrimers for targeting tumour cells expressing high-affinity folate receptor (hFR). These conjugates increased the longitudinal relaxation rate of tumour cells expressing the hFR, and subsequently the investigators demonstrated the specific targeting ability of folate–PAMAM dendrimer MRI contrast agents to ovarian tumour xenografts [14•]. Because of the increased amount of gadolinium-ion delivery per receptor using the dendrimer complex, the investigators have shown a 33% increase by contrast enhancement compared with that of a non-specific agent. More recent studies have been conducted by our group with non-ionic (surface amines capped with acetic anhydride) folateconjugated PAMAM dendrimers labeled with fluorescein isothiocyanate (FITC) for targeting tumour cells expressing hFR [15••]. Methotrexate and Taxol drug conjugates of these folate/FITC-conjugated PAMAM dendrimers (Figure 2a) are being investigated for their in vitro and in vivo cytotoxicity and specificity of drug targeting. Another approach to hFR targeting involves folic acid surface modification on polyarylether dendrons and dendrimers [16]. Folic acid is conjugated to the surface hydrazides by active ester formation and EDC coupling strategy. A similar reaction sequence was used to prepare a second-generation dendron-methotrexate conjugate. Unlike the folate–PAMAM dendrimer conjugates, which are water soluble, the folate–dendron conjugates prepared by convergent polyether linkages show turbidity near pH 7.4 [16]. Unfortunately, attempts to improve water solubility by the attachment of a polyethylene glycol (PEG) chain to the free hydroxyl group of these dendrons decreased the binding of folic acid and increased polydispersity. Approaches to engineer dendrimers with the ability to release drugs To improve biocompatibility and solubility, PAMAM dendrimers with PEG grafts on the surface have been synthesized, and the encapsulation of anticancer drugs, including adriamycin and methotrexate, using these structures has been attempted [17]. The ability to encapsulate the drugs in the dendritic core increases with increased generation and chain length of the attached PEG grafts. Though the drugs are released slowly from the matrix in low ionic strength aqueous solution, they are readily released in isotonic solutions. This suggests the need for further control of the drug release mechanism in these molecules. The pH-dependent change in the hydrodynamic radii of acid-terminated dendrimers has been investigated as a potential controlled-release system for encapsulated drugs from the interior hydrophobic areas of the dendrimer [18]. Using this principle, Paleos and co-workers [19] have investigated the pH-dependent inclusion and release of pyrene in quarternized poly(propylene imine) dendrimers (Figure 1d). The terminal quarternary ammonium salt not only enhances the water solubility of the dendrimer, but possess bactericidal, antifungal and antimicrobial properties. Pyrene is released when the internal tertiary amines get protonated between pH 4–2. This release within a Dendritic polymer macromolecular carriers for drug delivery Patri, Majoros and Baker narrower pH region suggests these materials are potential candidates for pH-sensitive controlled-release drugdelivery applications. Fréchet and co-workers [20,21] have prepared novel polyaryl ether dendrimers containing dual functionality on the surface by using a convergent synthetic strategy. One type of functionality is used to attach PEG units on the surface to render water solubility to the assembly, whereas the other functionality is utilized to attach hydrophobic drug molecules. They have conjugated cholesterol, pheynylalanine and tryptophan by carbonate, ester and carbamate linkages, respectively, and also synthesized a series of dendritic unimolecular micelles with a hydrophobic polyether core surrounded by a hydrophilic PEG shell for drug encapsulation [21]. A third-generation micelle with 24 surface chains has been shown to entrap the model drug indomethacin, loading up to an 11 wt% (approximately 9–10 drug molecules) per micelle. The in vitro drugrelease characteristics of these micelles were investigated and found to be slow and sustained, as compared with that of a cellulose membrane control. Together, these studies suggest that the physico-chemical structure of the dendrimer may allow for the loading and controlled release of drugs. Additional unique dendrimer-based macromolecules with delivery capability: immunoconjugates Antibodies are useful in targeted drug therapy because of the inherent specificity of the antibody–antigen interaction (Figure 2b). Modification of the antibody molecule with drugs often diminishes or eliminates its biological activity and reduces the targeting potential. An attractive approach to increase drug loading while retaining specificity is to link the antibody to another macromolecule, containing the drug, at a single site. Several research groups have taken this approach and employed dendrimers that carry imaging or therapeutic agents. Roberts et al. [22] have utilized dendrimers as linkers to covalently couple porphyrin to chelated copper ions. The resulting antibody PAMAM dendrimer conjugates retained 90% of the immunoreactivity of the unmodified antibody. They have also investigated the site of attachment of porphyrin in the conjugate and observed 100% of the conjugate bound to the heavy chain of the antibody. This is in contrast to a random-labeling technique in which only 69% of porphyrin was attached to the heavy chain and 31% to the light chain in the vicinity of antigen recognition. These radiolabeled antibody–dendrimer conjugates have potential application in cancer imaging and therapy. Barth and co-workers have prepared boronated starburst dendrimer–monoclonal-antibody immunoconjugates [23] for boron neutron capture therapy [24] (BNCT). They have compared the in vivo distribution pattern of 125I-labeled and boronated MoAb–dendrimer conjugates and reported that the dendrimers have a propensity to localize in the liver and spleen. Studies with non-boronated dendrimers revealed that this was directly 469 related to the molecular weight and number of terminal amino groups. The zero-generation starburst dendrimer with three terminal amino groups had the lowest hepatic and splenic uptake, with 1% and 0.01% respectively, of the injected dose of radioactivity at 72 h. Higher-generation (G2–G4) dendrimers have five times more hepatic uptake than the zero-generation dendrimer. PAMAM dendrimer–antibody conjugates have also been used to enhance the sensitivity of immunoassays [25,26], radioimmunotherapy [27] and imaging with minimal loss of immunoreactivity. A fifth-generation PAMAM dendrimer labeled with FITC, capped with acetic anhydride to neutralize surface amines and minimize non-specific interaction with the cell surface, was conjugated [28] to anti-PSMA (prostate-specific membrane antigen) antibody for targeting prostate cancer. This conjugate has been shown to successfully target PSMA-positive LNCaP cell line with minimal loss of immunoreactivity, compared with the control without the receptor. Further investigation for the drug-delivery and radioisotope studies are underway at our laboratories. Dendrimers clustered around a central core molecule, also described as tectodendrimers, are prepared using dendrimers as core and shell reagents [29••]. This type of cluster reagent (Figure 2c) has been prepared with fluorescein in the core reagent for detection, and folate moieties in the shell reagent for targeting. This minimizes the solubility problems encountered in previous studies with aromatic FITC moieties on the surface of the dendrimer while maximizing the surface availability of the targeting agent. This molecule has successfully targeted hFR cell lines and appears potentially more efficient than a single dendrimer having both agents on the surface of the polymer. Specific studies on the safety and efficiency of dendrimer-mediated drug delivery The true utility determination of drug-delivery agents requires testing in appropriate animal models. Recent studies have begun this difficult process. Duncan and coworkers [30••] have investigated the relationship between structure and biocompatibility of PAMAM, poly(propyleneimine), poly(ethylene oxide) grafted carbosilane dendrimers with cationic (NH2-terminated) and anionic (COONa-terminated) dendrimers in vitro. They have reported that, regardless of structure, cationic dendrimers were generally haemolytic and cytotoxic at even relatively low concentrations. In the case of PAMAM dendrimers, haemolysis is generation dependent (generations 1–4) with it increased in higher generations. Conversely, dendrimers with carboxylate-terminal groups were neither haemolytic nor did they cause cytotoxicity of a panel of cell lines studied in vitro. They have further observed that PAMAM dendrimers of equivalent surface functionality were slightly less toxic than DAB (polypropylenimine) dendrimers with the same number of surface groups. They have also investigated the biodistribution of 125I-labelled PAMAM dendrimers in vivo and reported that cationic dendrimers 470 Next-generation therapeutics were cleared rapidly from the circulation after intravenous and intraperitoneal administration. Anionic PAMAM dendrimers showed longer circulation times with generation-dependent clearance rates, the lower generations circulating longer. Using an everted rat intestinal sac system, Duncan and coworkers [31] have studied the effect of PAMAM dendrimer size, charge and concentration on uptake and transport across adult rat intestine in vitro. The results obtained from this study show that 125I-labeled anionic dendrimers have rapid serosal transfer rates and low tissue deposition. The size or conformation sensitivity of the transport mechanism was indicated as a generation 5.5 dendrimer displaying higher tissue accumulation compared with that of either a generation 2.5 or 3.5 polymer. In contrast to this, cationic PAMAM dendrimers were associated with lower transport rates as the negatively charged cell membranes appeared to interact with the cationic dendrimer surface. Ghandehari et al. [32] have investigated the influence of increase in size and molecular weight of fluorescentlabeled PAMAM dendrimers on the extravasation across microvascular endothelium and compared these molecules with corresponding linear poly(ethylene glycol) of similar molecular weight. They have observed an exponential increase in the extravasation time with an increase in the molecular weight and size of PAMAM dendrimers, from generation zero through generation four, which increased from 143.9 s to 422.7 s. It appeared that as the molecular diameter and size of the PAMAM dendrimer increases with generation, the exerted viscous drag on the polymer together with its degree of exclusion from the endothelial pores increases, and hence the observed increase in the extravasation time. Compared with PAMAM dendrimers, PEG molecules with a similar molecular weight took a longer time to extravasate across the endothelium into the interstitial tissue. For example, a 6.0 kDa PEG had a higher extravasation time of 453.9 s compared with that of the similar molecular weight third-generation (molecular weight 6909) with 203.8 s. It is proposed that this is the result of a larger hydrodynamic volume on the hydrated PEG chain because of the coiled conformation. The permeability of a series of PAMAM dendrimers across MDCK (Madin–Darby canine kidney) cell line also has been investigated [33]. The investigators labeled G0–G4 PAMAMs with FITC; and determined the permeability order was G4>> G1 ≈ G0 > G3 > G2. This suggested that although dendrimers traverse the vascular endothelium, there might be a size threshold for this migration. Conclusions Although dendrimer drug-delivery is in its infancy, it offers several attractive features. It provides a uniform platform for drug attachment that has the ability to bind and release drugs through several mechanisms. Although toxicity problems may exist, modification of the structure should resolve these issues. Further work is needed to define the structure of the polymer and the relationship between the polymer and drug molecules for this technology to succeed in drug delivery. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1. Pillai O, Panchagnula R: Polymers in drug delivery. Curr Opin Chem Biol 2001, 5:447-451. 2. Fréchet JMJ, Tomalia DA: Dendrimers and other Dendritic Polymers. •• Chichester, UK: Wiley; 2002. Please refer to these recently published books (see also [3••]), edited/written by the pioneers in the field of dendrimers, for a comprehensive review of this field. 3. •• Newkome GR, Moorefield CN, Vögtle F: Dendrimers and Dendrons: Concepts, Syntheses, Applications. Weinheim, Germany: Wiley-VCH; 2001. See annotation to [2••]. 4. Tomalia DA, Baker H, Dewald JR, Hall M, Kallos G, Martin S, Roeck J, Ryder J, Smith P: A new class of polymers: starburst-dendritic macromolecules. Polym J (Tokyo) 1985, 17:117-132. 5. Newkome GR, Yao Z, Baker GR, Gupta VK: Cascade molecules: a new approach to micelles. A [27]-Arborol. J Org Chem 1985, 50:2003-2004. 6. Jansen JFGA, de Brabander-van den Berg EMM, Meijer EW: Encapsulation of guest molecules into a dendritic box. Science 1994, 266:1226-1229. 7. Esfand R, Tomalia DA: Poly(amidoamine) (PAMAM) dendrimers: from biomimicry to drug delivery and biomedical applications. Drug Discov Today 2001, 6:427-436. 8. 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Konda SD, Aref M, Wang S, Brechbiel M, Wiener EC: Specific • targeting of folate-dendrimer MRI contrast agents to the high affinity folate receptor expressed in ovarian tumor xenogafts. Magn Reson Mater Phys Biol Med 2001, 12:104-113. Wiener’s group is the first to use the folate-conjugated PAMAM dendrimers for specific targeting to use in MRI contrast agents. This paper describes the use of such a device in targeting ovarian tumor and contrast enhancement in vivo. 15. Baker JR Jr, Quintana A, Piehler L, Banazak-Holl M, Tomalia DA, •• Raczka E: The synthesis and testing of anti-cancer therapeutic nanodevices. Biomed Microdevices 2001, 3:61-69. This paper describes the use of dendrimers as a novel therapeutic nanodevice for targeting, imaging and drug delivery to the cancer cells. 16. Kono K, Liu M, Fréchet JMJ: Design of dendritic macromolecules containing folate or methotrexate residues. Bioconjugate Chem 1999, 10:1115-1121. 17. 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Malik N, Wiwattanapatapee R, Klopsch R, Lorenz K, Frey H, •• Weener JW, Meijer EW, Paulus W, Duncan R: Dendrimers: relationship between structure and biocompatibility in vitro, and preliminary studies on the biodistribution of 125I-labelled polyamidoamine dendrimers in vivo. J Controlled Release 2000, 65:133-148. This paper describes the in vitro and in vivo biodistribution studies of PAMAM dendrimers. It is essential to understand the effect of size, surface charge and functionality of dendrimers in vivo to ascertain their biocompatibility and toxicity for drug-delivery applications. 24. Soloway AH, Tjarks W, Barnum BA, Rong F-G, Barth RF, Codogni IM, Wilson JG: The chemistry of neutron capture therapy. Chem Rev 1998, 98:1515-1562. 31. Wiwattanapatapee R, Carreño-Gómez B, Malik N, Duncan R: Anionic PAMAM dendrimers rapidly cross adult rat intestine in vitro: a potential oral delivery system? Pharm Res 2000, 17:991-998. 25. Singh P, Moll F III, Lin SH, Ferzli C: Starburst dendrimers: a novel matrix for multifunctional reagents in immunoassays. Clin Chem 1996, 42:1567-1569. 32. El-Sayed M, Kiani MF, Naimark MD, Hikal AH, Ghandehari H: Extravasation of poly(amidoamine) (PAMAM) dendrimers across microvascular network endothelium. Pharm Res 2001, 18:23-28. 26. Ong KK, Jenkins AL, Cheng R, Tomalia DA, Durst HD, Jensen JL, Emanuel PA, Swim CR, Yin R: Dendrimer enhanced immunosensors for biological detection. Anal Chim Acta 2001, 444:143-148. 33. Tajarobi F, El-Sayed M, Rege BD, Polli JE, Ghandehari H: Transport of poly amidoamine dendrimers across Madin–Darby canine kidney cells. Int J Pharm 2001, 215:263-267. 22. Roberts JC, Adams YE, Tomalia D, Mercer-Smith JA, Lavallee DK: Using starburst dendrimers as linker molecules to radiolabel antibodies. Bioconjugate Chem 1990, 1:305-308.