24 research outputs found
Synthesis and Functional Evaluation of DNA-Assembled Polyamidoamine Dendrimer Clusters for Cancer Cell-Specific Targeting
SummaryWe sought to produce dendrimers conjugated to different biofunctional moieties (fluorescein [FITC] and folic acid [FA]), and then link them together using complementary DNA oligonucleotides to produce clustered molecules that target cancer cells that overexpress the high-affinity folate receptor. Amine-terminated, generation 5 polyamidoamine (G5 PAMAM) dendrimers are first partially acetylated and then conjugated with FITC or FA, followed by the covalent attachment of complementary, 5′-phosphate-modified 34-base-long oligonucleotides. Hybridization of these oligonucleotide conjugates led to the self-assembly of the FITC- and FA-conjugated dendrimers. In vitro studies of the DNA-linked dendrimer clusters indicated specific binding to KB cells expressing the folate receptor. Confocal microscopy also showed the internalization of the dendrimer cluster. These results demonstrate the ability to design and produce supramolecular arrays of dendrimers using oligonucleotide bridges. This will also allow for further development of DNA-linked dendrimer clusters as imaging agents and therapeutics
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<sub><i>m</i></sub>-(Es)MTX<sub><i>n</i></sub> and G5-GLA<sub><i>m</i></sub>-(Am)MTX<sub><i>n</i></sub>, respectively.
In serum-containing medium, free MTX was slowly released from “G5-GLA<sub><i>m</i></sub>-(Es)MTX<sub><i>n</i></sub>”,
with ∼8% MTX released from the dendrimer in 72 h, whereas the
MTX on G5-GLA<sub><i>m</i></sub>-(Am)MTX<sub><i>n</i></sub> was completely stable. The G5-GLA<sub><i>m</i></sub>-(Am)MTX<sub><i>n</i></sub> 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<sub><i>m</i></sub>-MTX<sub><i>n</i></sub> may serve as a serum-stable
nanoparticle conjugate to specifically and effectively target and
treat PSMA-overexpressing prostate tumors
HER2 specific delivery of methotrexate by dendrimer conjugated anti-HER2 mAb
"Herceptin, a humanized monoclonal antibody that binds to human growth factor receptor-2 (HER2), was covalently attached to a fifth-generation (G5) polyamidoamine dendrimer containing the cytotoxic drug methotrexate. The specific binding and internalization of this conjugate labeled with FITC was clearly demonstrated in cell lines overexpressing HER2 by flow cytometry as well as confocal microscopic analysis. In addition, binding and uptake of antibody conjugated dendrimers was completely blocked by excess non-conjugated herceptin. The dendrimer conjugate was also shown to inhibit the dihydrofolate reductase with similar activity to methotrexate. Co-localization experiments with lysotracker red indicate that antibody conjugate, although internalized efficiently into cells, has an unusually long residence time in the lysosome. Somewhat lower cytotoxicity of the conjugate in comparison to free methotrexate was attributed to the slow release of methotrexate from the conjugate and its long retention in the lysosomal pocket."http://deepblue.lib.umich.edu/bitstream/2027.42/64148/1/nano8_29_295102.pd
Specific and Cooperative Interactions between Oximes and PAMAM Dendrimers As Demonstrated by <sup>1</sup>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, <sup>1</sup>H–<sup>1</sup>H 2D spectroscopy (COSY, NOESY), and <sup>1</sup>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
Association of Variants in TMEM45A With Keratoglobus
IMPORTANCE: Keratoglobus is a rare corneal disorder characterized by generalized thinning and globular protrusion of the cornea. Affected individuals typically have significantly decreased vision and are at risk of corneal perforation. The genetic basis and inheritance pattern of isolated congenital keratoglobus are currently unknown. OBJECTIVE: To identify the genetic basis of isolated congenital keratoglobus. DESIGN, SETTING, AND PARTICIPANTS: This case series and molecular analysis studied 3 unrelated nonconsanguineous families with keratoglobus at a medical center in Israel. Data were collected from June 2019 to March 2021 and analyzed during the same period. EXPOSURES: Whole-exome sequencing and direct Sanger sequencing, expression analysis by real-time polymerase chain reaction, splice-site variant analysis, immunohistochemical staining, and histological evaluation of a knockout mouse model. MAIN OUTCOMES AND MEASURE: Molecular characteristics associated with keratoglobus. RESULTS: Four pediatric patients (3 male individuals) from 3 families had clinical findings consistent with keratoglobus. These included globular protrusion, corneal thinning more prominent at the periphery, and high astigmatism. Truncating and splice site variants were identified in the TMEM45A gene, which fully segregate with the disorder. All affected individuals were homozygous or compound heterozygous for variants in the TMEM45A gene, while unaffected family members were heterozygous carriers. Expression analysis in healthy controls showed that TMEM45A was expressed 23 times higher in the human cornea compared with peripheral blood. Immunohistochemical staining of the TMEM45A protein in normal corneas confirmed its expression in the corneal stroma and epithelium. A TMEM45A knockout mouse model showed structural features consistent with keratoglobus. CONCLUSIONS AND RELEVANCE: Expression of TMEM45A has been previously shown to result in upregulation of extracellular matrix components and fibrosis. These results suggest that isolated congenital keratoglobus is an autosomal recessively inherited disorder associated with variants in the TMEM45A gene
The synthesis of control (GdIII-DOTA-G5) and FA targeted dendritic chelate (GdIII-DOTA-G5-FA)
After conjugation of 4.5 (on average) of folic acid molecules to G5 PAMAM dendrimer (), 50 of the primary amine groups were acetylated (), the remaining primary amines conjugated with bifunctional NCS-DOTA () and complexed with GdCl.6HO (). The structure of the DOTA-NCS used for conjugation of contrast agents (lower panel).<p><b>Copyright information:</b></p><p>Taken from "Targeted gadolinium-loaded dendrimer nanoparticles for tumor-specific magnetic resonance contrast enhancement"</p><p></p><p>International Journal of Nanomedicine 2008;3(2):201-210.</p><p>Published online Jan 2008</p><p>PMCID:PMC2527674.</p><p>© 2008 Swanson et al, publisher and licensee Dove Medical Press Ltd.</p
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<sub><i>n</i></sub> 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<sub>10</sub> 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<sub><i>n</i></sub> conjugate may serve as a FR-targeted chemotherapeutic
with potential for cancer therapy