Asymmetrical flow field-flow fractionation for improved characterization of human plasma lipoproteins.

High- and low-density lipoproteins (HDL and LDL) are attractive targets for biomarker discovery. However, ultracentrifugation (UC), the current methodology of choice for isolating HDL and LDL, is tedious, requires large sample volume, results in sample loss, and does not readily provide information on particle size. In this work, human plasma HDL and LDL are separated and collected using semi-preparative asymmetrical flow field-flow fractionation (SP-AF4) and UC. The SP-AF4 and UC separation conditions, sample throughput, and liquid chromatography/mass spectrometry (LC/MS) lipidomic results are compared. Over 600 µg of total proteins is recovered in a single SP-AF4 run, and Western blot results confirm apoA1 pure and apoB100 pure fractions, consistent with HDL and LDL, respectively. The SP-AF4 separation requires ~ 60 min per sample, thus providing a marked improvement over UC which can span hours to days. Lipidome analysis of SP-AF4-prepared HDL and LDL fractions is compared to UC-prepared HDL and LDL samples. Over 270 lipids in positive MS mode and over 140 lipids in negative MS mode are identified by both sample preparation techniques with over 98% overlap between the lipidome. Additionally, lipoprotein size distributions are determined using analytical scale AF4 coupled with multiangle light scattering (MALS) and dynamic light scattering (DLS) detectors. These developments position SP-AF4 as a sample preparation method of choice for lipoprotein biomarker characterization and identification. Graphical abstract ᅟ.

Folate Receptor-Targeted Dendrimer-Methotrexate Conjugate for Inflammatory Arthritis.

Generation 5 (G5) poly(amidoamide) (PAMAM) dendrimers are synthetic polymers that have been broadly applied as drug delivery carriers. Methotrexate (MTX), an anti-folate metabolite, has been successfully used as an anti-inflammatory drug to treat rheumatoid arthritis (RA) in the clinic. In this study, we examine the therapeutic efficacy of G5 PAMAM dendrimer methotrexate conjugates (G5-MTX) that also have folic acid (FA) conjugated to the G5-MTX (G5-FA-MTX) to target inflammation-activated folate receptors overexpressing macrophages. These cells are thought to play an important role in the development of RA. With G5 serving as a control, the in vitro binding affinities of G5-FA-MTX and G5-MTX to activated macrophages were assessed in RAW264.7, NR8383 and primary rat peritoneal macrophages. The results indicated that the binding of either conjugate to macrophages was concentration- and temperature-dependent and could be blocked by the presence of 6.25 mM free FA (p < 0.005). The preventive effects of G5-MTX and G5-FA-MTX conjugates on the development of arthritis were explored on an adjuvant-induced inflammatory arthritis model and had similar preventive effects in inflammatory arthritis at a MTX equivalent dose of 4.95 µmol/kg. These studies indicated that when multiples of MTX are conjugated on dendritic polymers, they specifically bind to folate receptor overexpressing macrophages and have comparable anti-inflammatory effects to folate targeted MTX conjugated polymers.

PSMA-targeted stably linked "dendrimer-glutamate urea-methotrexate" as a prostate cancer therapeutic.

One of the important criteria for achieving efficient nanoparticle-based targeted drug delivery is that the drug is not prematurely released at off-target sites. Here we report the preclinical evaluation of a serum-stable dendrimer-based drug conjugate capable of actively targeting into prostate cancer (PC) cells, delivered through the prostate-specific membrane antigen (PSMA). Multiple molecules of PSMA-binding small molecule glutamate urea (GLA; targeting agent) and the drug methotrexate (MTX) were conjugated to generation 5 PAMAM dendrimer (G5) through Cu-free "click" chemistry. The GLA was conjugated through a stable amide bond, and the MTX was conjugated either through ester (Es)- or amide (Am)-coupling, to generate G5-GLA(m)-(Es)MTX(n) and G5-GLA(m)-(Am)MTX(n), respectively. In serum-containing medium, free MTX was slowly released from "G5-GLA(m)-(Es)MTX(n)", with ~8% MTX released from the dendrimer in 72 h, whereas the MTX on G5-GLA(m)-(Am)MTX(n) was completely stable. The G5-GLA(m)-(Am)MTX(n) bound and internalized into PSMA-expressing LNCaP cells, but not into PSMA-negative PC3 cells. The conjugate-inhibited recombinant dihydrofolate reductase and induced potent cytotoxicity in the LNCaP cells, but not in the PC3 cells. Similar to the action of free GLA, stable amide-linked dendrimer-GLA was capable of inhibiting the enzyme N-acetylated α-linked acidic dipeptidase (NAALADase) activity of PSMA. The G5-GLA(m)-MTX(n) may serve as a serum-stable nanoparticle conjugate to specifically and effectively target and treat PSMA-overexpressing prostate tumors.

Design and mechanistic investigation of oxime-conjugated PAMAM dendrimers as the catalytic scavenger of reactive organophosphate.

Pyridinium aldoxime (PAM) molecules constitute a group of small molecule antidotes essential for the treatment of reactive organophosphate (OP) poisoning. Their therapeutic efficacy stems from a combination of two activities: (i) reactivation of acetylcholine esterase inhibited by OP; (ii) scavenging of free OP. Here, we report the design, synthesis and in vitro functional characterization of fifth generation poly(amidoamine)dendrimer conjugates, each tethered with PAM or hydroxamate, as OP scavenging macromolecules. We chose paraoxon (POX) as the model OP, and performed extensive reaction kinetic studies in aqueous media to determine the activities and mechanisms of these dendrimer conjugates in POX hydrolysis with 1H NMR, UV-vis and LCMS/MS methods. Quantitative kinetic analysis suggests that the conjugate hydrolyzed POX through a catalytic mechanism as effective as that of the unconjugated molecules. In summary, we report the first class of dendrimer conjugates which are stable in the plasma and functioning as OP-responsive catalytic scavengers.

Avidity modulation of folate-targeted multivalent dendrimers for evaluating biophysical models of cancer targeting nanoparticles.

We investigated two types of generation 5 polyamidoamine (PAMAM) dendrimers, each conjugated stochastically with a mean number of 5 or 10 methotrexate (MTX) ligands per dendrimer (G5-MTX5, G5-MTX10), for their binding to surface-immobilized folate binding protein (FBP) as a function of receptor density. The binding study was performed under flow by surface plasmon resonance spectroscopy. Two multivalent models were examined to simulate binding of the dendrimer to the receptor surface, showing that at relatively high receptor density, both dendrimer conjugates exhibit high avidity. However, upon reducing the receptor density by a factor of 3 and 13 relative to the high density level, the avidity of the lower-valent G5-MTX5 decreases by up to several orders of magnitude (KD = nM to µM), whereas the avidity of G5-MTX10 remains largely unaffected regardless of the density variation. Notably, on the 13-fold reduced FBP surface, G5-MTX5 displays binding kinetics similar to that of monovalent methotrexate, which is patently different from the still tight binding of the higher-valent G5-MTX10. Thus, the binding analysis demonstrates that avidity displayed by multivalent MTX conjugates varies in response to the receptor density and can be modulated for achieving tighter, more specific binding to the higher receptor density by modulation of ligand valency. We believe this study provides experimental evidence supportive of the mechanistic hypothesis of multivalent NP uptake to a cancer cell over a healthy cell where the diseased cell expresses the folate receptor at higher density.

Design and in vitro validation of multivalent dendrimer methotrexates as a folate-targeting anticancer therapeutic.

Design of cancer-targeting nanotherapeutics relies on a pair of two functionally orthogonal molecules, one serving as a cancer cell-specific targeting ligand, and the other as a therapeutic cytotoxic agent. The present study investigates the validity of an alternative simplified strategy where a dual-acting molecule which bears both targeting and cytotoxic activity is conjugated to the nanoparticle as cancer-targeting nanotherapeutics. Herein, we demonstrate that methotrexate is applicable for this dual-acting strategy due to its reasonable affinity to folic acid receptor (FAR) as a tumor biomarker, and cytotoxic inhibitory activity of cytosolic dihydrofolate reductase. This article describes design of new methotrexate-conjugated poly(amidoamine) (PAMAM) dendrimers, each carrying multiple copies of methotrexate attached through a stable amide linker. We evaluated their dual biological activities by performing surface plasmon resonance spectroscopy, a cell-free enzyme assay and cell-based experiments in FAR-overexpressing cells. This study identifies the combination of an optimal linker framework and multivalency as the two key design elements that contribute to achieving potent dual activity.

Dendrimer-based multivalent vancomycin nanoplatform for targeting the drug-resistant bacterial surface.

Vancomycin represents the preferred ligand for bacteria-targeting nanosystems. However, it is inefficient for emerging vancomycin-resistant species because of its poor affinity to the reprogrammed cell wall structure. This study demonstrates the use of a multivalent strategy as an effective way for overcoming such an affinity limitation in bacteria targeting. We designed a series of fifth generation (G5) poly(amidoamine) (PAMAM) dendrimers tethered with vancomycin at the C-terminus at different valencies. We performed surface plasmon resonance (SPR) studies to determine their binding avidity to two cell wall models, each made with either a vancomycin-susceptible (D)-Ala-(D)-Ala or vancomycin-resistant (D)-Ala-(D)-Lac cell wall precursor. These conjugates showed remarkable enhancement in avidity in the cell wall models tested, including the vancomycin-resistant model, which had an increase in avidity of four to five orders of magnitude greater than free vancomycin. The tight adsorption of the conjugate to the model surface corresponded with its ability to bind vancomycin-susceptible Staphylococcus aureus bacterial cells in vitro as imaged by confocal fluorescent microscopy. This vancomycin platform was then used to fabricate the surface of iron oxide nanoparticles by coating them with the dendrimer conjugates, and the resulting dendrimer-covered magnetic nanoparticles were demonstrated to rapidly sequester bacterial cells. In summary, this article investigates the biophysical basis of the tight, multivalent association of dendrimer-based vancomycin conjugates to the bacterial cell wall, and proposes a potential new use of this nanoplatform in targeting Gram-positive bacteria.

Sustained analgesia achieved through esterase-activated morphine prodrugs complexed with PAMAM dendrimer.

PURPOSE: Design and evaluate the in vitro and in vivo efficacy of two extended release morphine formulations developed for IV administration by complexing esterase activated morphine prodrugs to surface-modified, generation 5 (G5) poly(amidoamine) (PAMAM) dendrimer. METHODS: Prodrugs were synthesized, complexed with PAMAM dendrimer, characterized via ultra performance liquid chromatography (UPLC), nuclear magnatic resonance (NMR), and tested in vitro using rat plasma vs. saline control and in an in vivo rat and guinea pig pain model (modified Randall and Selitto test). RESULTS: We demonstrated that complexation with dendrimer allowed the solubilization of the prodrugs for in vivo applications without the need for salt, and that the structural design of the morphine prodrugs allowed the controlled release of morphine which extended the action of morphine-induced analgesia in an animal pain model from 2 h (control) to 6 h (Morphine Prodrug A). CONCLUSION: The concept of complexing/solubilizing appropriately designed esterase-sensitive prodrugs with dendrimer to enhance the sustained release of these drugs may be a useful pharmacokinetic strategy for a range of therapeutics.

Specific and cooperative interactions between oximes and PAMAM dendrimers as demonstrated by (1)H NMR study.

Oximes are important in the treatment of organophosphate (OP) poisoning, but have limited biological half-lives. Complexing these drugs with a macromolecule, such as a dendrimer, could improve their pharmacokinetics. The present study investigates the intermolecular interactions that drive the complexation of oxime-based drug molecules with fifth generation poly(amidoamine) (PAMAM) dendrimers. We performed steady-state binding studies of two molecules, pralidoxime and obidoxime, employing multiple NMR methods, including 1D titration, (1)H-(1)H 2D spectroscopy (COSY, NOESY), and (1)H diffusion-ordered spectroscopy (DOSY). Several important insights were gained in understanding the host-guest interactions occurring between the drug molecules and the polymer. First, the guest molecules bind to the dendrimer macromolecule through a specific interaction rather than through random, hydrophobic encapsulation. Second, this specificity is driven primarily by the electrostatic or H-bond interaction of the oxime at a dendrimer amine site. Also, the average strength for each drug and dendrimer interaction is affected by the surface modification of the polymer. Third, individual binding events between oximes and a dendrimer have a negative cooperative effect on subsequent oxime binding. In summary, this report provides a novel perspective important for designing host systems for drug delivery.

Polyvalent dendrimer-methotrexate as a folate receptor-targeted cancer therapeutic.

Our previous studies have demonstrated that a generation 5 dendrimer (G5) conjugated with both folic acid (FA) and methotrexate (MTX) has a higher chemotherapeutic index than MTX alone. Despite this, batch-to-batch inconsistencies in the number of FA and MTX molecules linked to each dendrimer led to conjugate batches with varying biological activity, especially when scaleup synthesis was attempted. Since the MTX is conjugated through an ester linkage, there were concerns that biological inconsistency could also result from serum esterase activity and differential bioavailability of the targeted conjugate. In order to resolve these problems, we undertook a novel approach to synthesize a polyvalent G5-MTX(n) conjugate through click chemistry, attaching the MTX to the dendrimer through an esterase-stable amide linkage. Surface plasmon resonance binding studies show that a G5-MTX(10) conjugate synthesized in this manner binds to the FA receptor (FR) through polyvalent interaction showing 4300-fold higher affinity than free MTX. The conjugate inhibits dihydrofolate reductase, and induces cytotoxicity in FR-expressing KB cells through FR-specific cellular internalization. Thus, the polyvalent MTX on the dendrimer serves the dual role as a targeting molecule as well as a chemotherapeutic drug. The newly synthesized G5-MTX(n) conjugate may serve as a FR-targeted chemotherapeutic with potential for cancer therapy.

Optical fiber-based in vivo quantification of growth factor receptors.

BACKGROUND: Growth factor receptors such as epidermal growth factor receptor 1 and human epidermal growth receptor 2 (HER2) are overexpressed in certain cancer cells. Antibodies against these receptors (eg. cetuximab and transtuzumab [Herceptin]) have shown therapeutic value in cancer treatment. The existing methods for the quantification of these receptors in tumors involve immunohistochemistry or DNA quantification, both in extracted tissue samples. The goal of the study was to evaluate whether an optical fiber-based technique can be used to quantify the expression of multiple growth factor receptors simultaneously. METHODS: The authors examined HER2 expression using the monoclonal antibody trastuzumab as a targeting ligand to test their system. They conjugated trastuzumab to 2 different Alexa Fluor dyes with different excitation and emission wavelengths. Two of the dye conjugates were subsequently injected intravenously into mice bearing HER2-expressing subcutaneous tumors. An optical fiber was then inserted into the tumor through a 30-gauge needle, and using a single laser beam as the excitation source, the fluorescence emitted by the 2 conjugates was identified and quantified by 2-photon optical fiber fluorescence. RESULTS: The 2 conjugates bound to the HER2-expressing tumor competitively in a receptor-specific fashion, but they failed to bind to a similar cell tumor that did not express HER2. The concentration of the conjugate present in the tumor as determined by 2-photon optical fiber fluorescence was shown to serve as an index of the HER2 expression levels. CONCLUSIONS: These studies offer a minimally invasive technique for the quantification of tumor receptors simultaneously.

Bifunctional PAMAM dendrimer conjugates of folic acid and methotrexate with defined ratio.

Our group previously developed a multifunctional, targeted cancer therapeutic based on Generation 5 (G5) polyamidoamine (PAMAM) dendrimers. In those studies we conjugated the targeting molecule folic acid (FA) and the chemotherapeutic drug methotrexate (MTX) sequentially. This complex macromolecule was shown to selectively bind and kill KB tumor cells that overexpress folate receptor (FR) in vitro and in vivo. However, the multistep conjugation strategy employed in the synthesis of the molecule resulted in heterogeneous populations having differing numbers and ratios of the functionally antagonistic FA and MTX. This led to inconsistent and sometimes biologically inactive batches of molecules, especially during large-scale synthesis. We here resolved this issue by using a novel triazine scaffold approach that reduces the number of dendrimer conjugation steps required and allows for the synthesis of G5 conjugates with defined ratios of FA and MTX. Although an unoccupied γ-glutamyl carboxylate of FA has been previously suggested to be nonessential for FR binding, the functional requirement of an open α-carboxylate still remains unclear. In an attempt to also address this question, we have synthesized isomeric FA dendrimer conjugates (α-carboxyl or γ-carboxyl linked). Competitive binding studies revealed that both linkages have virtually identical affinity toward FR on KB cells. Our studies show that a novel bifunctional triazine-based conjugate G5-Triazine-γMTX-αFA with identical numbers of FA and MTX binds to FR through a polyvalent interaction and induces cytotoxicity in KB cells through FR-mediated cellular internalization, inducing higher toxicity as compared to conjugates synthesized by the multistep strategy. This work serves as a proof of concept for the development of bifunctional dendrimer conjugates that require a defined ratio of two functional molecules.

Photochemical release of methotrexate from folate receptor-targeting PAMAM dendrimer nanoconjugate.

Nanoparticle (NP)-based targeted drug delivery involves cell-specific targeting followed by a subsequent therapeutic action from the therapeutic carried by the NP system. NPs conjugated with methotrexate (MTX), a potent inhibitor of dihydrofolate reductase (DHFR) localized in cytosol, have been under investigation as a delivery system to target cancer cells to enhance the therapeutic index of methotrexate, which is otherwise non-selectively cytotoxic. Despite improved therapeutic activity from MTX-conjugated NPs in vitro and in vivo, the therapeutic action of these conjugates following cellular entry is poorly understood; in particular it is unclear whether the therapeutic activity requires release of the MTX. This study investigates whether MTX must be released from a nanoparticle in order to achieve the therapeutic activity. We report herein light-controlled release of methotrexate from a dendrimer-based conjugate and provide evidence suggesting that MTX still attached to the nanoconjugate system is fully able to inhibit the activity of its enzyme target and the growth of cancer cells.

Dendrimer-based multivalent methotrexates as dual acting nanoconjugates for cancer cell targeting.

Cancer-targeting drug delivery can be based on the rational design of a therapeutic platform. This approach is typically achieved by the functionalization of a nanoparticle with two distinct types of molecules, a targeting ligand specific for a cancer cell, and a cytotoxic molecule to kill the cell. The present study aims to evaluate the validity of an alternative simplified approach in the design of cancer-targeting nanotherapeutics: conjugating a single type of molecule with dual activities to nanoparticles, instead of coupling a pair of orthogonal molecules. Herein we investigate whether this strategy can be validated by its application to methotrexate, a dual-acting small molecule that shows cytotoxicity because of its potent inhibitory activity against dihydrofolate reductase and that binds folic acid receptor, a tumor biomarker frequently upregulated on the cancer cell surface. This article describes a series of dendrimer conjugates derived from a generation 5 polyamidoamine (G5 PAMAM) presenting a multivalent array of methotrexate and also demonstrates their dual biological activities by surface plasmon resonance spectroscopy, a cell-free enzyme assay, and cell-based experiments with KB cancer cells.

Folate-targeted nanoparticles show efficacy in the treatment of inflammatory arthritis.

OBJECTIVE: To investigate the uptake of a poly(amidoamine) dendrimer (generation 5 [G5]) nanoparticle covalently conjugated to polyvalent folic acid (FA) as the targeting ligand into macrophages, and to investigate the activity of an FA- and methotrexate (MTX)-conjugated dendrimer (G5-FA-MTX) as a therapeutic for the inflammatory disease of arthritis. METHODS: In vitro studies were performed in macrophage cell lines and in isolated mouse macrophages to check the cellular uptake of fluorescence-tagged G5-FA nanoparticles, using flow cytometry and confocal microscopy. In vivo studies were conducted in a rat model of collagen-induced arthritis to evaluate the therapeutic potential of G5-FA-MTX. RESULTS: Folate-targeted dendrimer bound and internalized in a receptor-specific manner into both folate receptor ß-expressing macrophage cell lines and primary mouse macrophages. The conjugate G5-FA-MTX acted as a potent antiinflammatory agent and reduced arthritis-induced parameters of inflammation such as ankle swelling, paw volume, cartilage damage, bone resorption, and body weight decrease. CONCLUSION: The use of folate-targeted nanoparticles to specifically target MTX into macrophages may provide an effective clinical approach for antiinflammatory therapy in rheumatoid arthritis.

Polyvalent saccharide-functionalized generation 3 poly(amidoamine) dendrimer-methotrexate conjugate as a potential anticancer agent.

A saccharide-terminated generation 3 (G3) polyamidoamine (PAMAM) dendrimer was synthesized as a drug carrier. Utilizing this dendritic platform, we have successfully synthesized polyvalent conjugates (G3-MTX) containing the drug methotrexate (MTX). Surface Plasmon Resonance (SPR) results showed that G3-MTX presented three orders of magnitude enhancement in binding avidity to folate-binding protein (FBP) as compared to the free folic acid (FA). Flow cytometric and confocal microscopic analysis showed that conjugate (G3-MTX-FI) containing imaging agent fluorescein-5(6)-carboxamidohexanoic acid (FI) was internalized into folate receptor (FR)-expressing KB cells in dose-dependent and receptor-mediated fashion. The G3-MTX induced a dose-dependent cytotoxicity in the KB cells. Therefore, the polyvalent G3-MTX may have potential as an anticancer nanodevice for the specific targeting and killing of FR-expressing tumor cells.

Design, synthesis, and biological functionality of a dendrimer-based modular drug delivery platform.

A modular dendrimer-based drug delivery platform was designed to improve upon existing limitations in single dendrimer systems. Using this modular strategy, a biologically active platform containing receptor mediated targeting and fluorescence imaging modules was synthesized by coupling a folic acid (FA) conjugated dendrimer with a fluorescein isothiocyanate (FITC) conjugated dendrimer. The two different dendrimer modules were coupled via the 1,3-dipolar cycloaddition reaction ("click" chemistry) between an alkyne moiety on the surface of the first dendrimer and an azide moiety on the second dendrimer. Two simplified model systems were also synthesized to develop appropriate "click" reaction conditions and aid in spectroscopic assignments. Conjugates were characterized by (1)H NMR spectroscopy and NOESY. The FA-FITC modular platform was evaluated in vitro with a human epithelial cancer cell line (KB) and found to specifically target the overexpressed folic acid receptor.

Plasma-mediated release of morphine from synthesized prodrugs.

Two morphine prodrugs ('PDA' and 'PDB') were synthesized and the kinetics of esterase-mediated morphine release from these prodrugs were determined when incubated with plasma from different animal species. Morphine was rapidly released from PDA by all species plasma with the maximum reached within 5-10min; the released morphine was biologically active as determined by an in vitro cAMP assay. The morphine was released from PDB at a slower and species-dependent rate (mouse>rat>guinea pig>human). Morphine's release from PDB appeared to be mediated by carboxyl esterases as the release was inhibited by the carboxyl esterase inhibitor benzil. PDA nor PDB induce cytotoxicity in the neuronal cell lines SK-NSH and SH-SY5Y. The carboxyl and amino functional moieties present on the linker portions of PDA and PDB, respectively, may facilitate their conjugation to nanoparticles to tailor morphine pharmacokinetics and specific targeting. These studies suggest the potential clinical utility of these prodrugs for morphine release at desired rates by administration of their mixture at selected ratios.

Fiber-optic multiphoton flow cytometry in whole blood and in vivo.

Circulating tumor cells in the bloodstream are sensitive indicators for metastasis and disease prognosis. Circulating cells have usually been monitored via extraction from blood, and more recently in vivo using free-space optics; however, long-term intravital monitoring of rare circulating cells remains a major challenge. We demonstrate the application of a two-photon-fluorescence optical fiber probe for the detection of cells in whole blood and in vivo. A double-clad fiber was used to enhance the detection sensitivity. Two-channel detection was employed to enable simultaneous measurement of multiple fluorescent markers. Because the fiber probe circumvents scattering and absorption from whole blood, the detected signal strength from fluorescent cells was found to be similar in phosphate-buffered saline (PBS) and in whole blood. The detection efficiency of cells labeled with the membrane-binding dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindoldicarbocyanine, 4-chlorobenzenesulfonate (DiD) was demonstrated to be the same in PBS and in whole blood. A high detection efficiency of green fluorescent protein (GFP)-expressing cells in whole blood was also demonstrated. To characterize in vivo detection, DiD-labeled untransfected and GFP-transfected cells were injected into live mice, and the cell circulation dynamics was monitored in real time. The detection efficiency of GFP-expressing cells in vivo was consistent with that observed ex vivo in whole blood.

Design of riboflavin-presenting PAMAM dendrimers as a new nanoplatform for cancer-targeted delivery.

This communication describes the synthesis and in vitro biological evaluation of novel generation 5 PAMAM dendrimers conjugated with riboflavin as a targeting ligand. Cell-based experiments demonstrated that a dendrimer conjugated with riboflavin is able to undergo cellular binding and uptake in KB cells, and when the dendrimer is also conjugated with methotrexate, the riboflavin dendrimer conjugate can potently inhibit cell growth.