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_<i>m</i>-(Es)­MTX_<i>n</i> and G5-GLA_<i>m</i>-(Am)­MTX_<i>n</i>, respectively. In serum-containing medium, free MTX was slowly released from “G5-GLA_<i>m</i>-(Es)­MTX_<i>n</i>”, with ∼8% MTX released from the dendrimer in 72 h, whereas the MTX on G5-GLA_<i>m</i>-(Am)­MTX_<i>n</i> was completely stable. The G5-GLA_<i>m</i>-(Am)­MTX_<i>n</i> 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_<i>m</i>-MTX_<i>n</i> may serve as a serum-stable nanoparticle conjugate to specifically and effectively target and treat PSMA-overexpressing prostate tumors

Specific and Cooperative Interactions between Oximes and PAMAM Dendrimers As Demonstrated by ¹H 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, ¹H–¹H 2D spectroscopy (COSY, NOESY), and ¹H 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_<i>n</i> 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–MTX₁₀ 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_<i>n</i> conjugate may serve as a FR-targeted chemotherapeutic with potential for cancer therapy

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 (<i>K</i>_D = 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

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