Anil K. Patri

DESIGN AND FUNCTION OF A DENDRIMER-BASED THERAPEUTIC NANODEVICE TARGETED TO TUMOR CELLS THROUGH THE FOLATE RECEPTOR

AbstractPurpose. We sought to develop nanoscale drug delivery materials that would allow targeted intracellular delivery while having an imaging capability for tracking uptake of the material. A complex nanodevice was designed and synthesized that targets tumor cells through the folate receptor.nMethods. The device is based on an ethylenediamine core polyamidoamine dendrimer of generation 5. Folic acid, fluorescein, and methotrexate were covalently attached to the surface to provide targeting, imaging, and intracellular drug delivery capabilities. Molecular modeling determined the optimal dendrimer surface modification for the function of the device and suggested a surface modification that improved targeting.nResults. Three nanodevices were synthesized. Experimental targeting data in KB cells confirmed the modeling predictions of specific and highly selective binding. Targeted delivery improved the cytotoxic response of the cells to methotrexate 100-fold over free drug.nConclusions. These results demonstrate the ability to design and produce polymer-based nanodevices for the intracellular targeting of drugs, imaging agents, and other materials.

Dendritic polymer macromolecular carriers for drug delivery

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.

Targeted gadolinium-loaded dendrimer nanoparticles for tumor-specific magnetic resonance contrast enhancement

A target-specific MRI contrast agent for tumor cells expressing high affinity folate receptor was synthesized using generation five (G5) of polyamidoamine (PAMAM) dendrimer. Surface modified dendrimer was functionalized for targeting with folic acid (FA) and the remaining terminal primary amines of the dendrimer were conjugated with the bifunctional NCS-DOTA chelator that forms stable complexes with gadolinium (Gd III). Dendrimer-DOTA conjugates were then complexed with GdCl3 followed by ICP-OES as well as MRI measurement of their longitudinal relaxivity (T1 s−1 mM−1) of water. In xenograft tumors established in immunodeficient (SCID) mice with KB human epithelial cancer cells expressing folate receptor (FAR), the 3D MRI results showed specific and statistically significant signal enhancement in tumors generated with targeted Gd(III)-DOTA-G5-FA compared with signal generated by non-targeted Gd(III)-DOTA-G5 contrast nanoparticle. The targeted dendrimer contrast nanoparticles infiltrated tumor and were retained in tumor cells up to 48 hours post-injection of targeted contrast nanoparticle. The presence of folic acid on the dendrimer resulted in specific delivery of the nanoparticle to tissues and xenograft tumor cells expressing folate receptor in vivo. We present the specificity of the dendrimer nanoparticles for targeted cancer imaging with the prolonged clearance time compared with the current clinically approved gadodiamide (Omniscan™) contrast agent. Potential application of this approach may include determination of the folate receptor status of tumors and monitoring of drug therapy.

Nanoparticle size and surface charge determine effects of PAMAM dendrimers on human platelets in vitro

Blood platelets are essential in maintaining hemostasis. Various materials can activate platelets and cause them to aggregate. Platelet aggregation in vitro is often used as a marker for materials thrombogenic properties, and studying nanomaterial interaction with platelets is an important step toward understanding their hematocompatibility. Here we report evaluation of 12 formulations of PAMAM dendrimers varying in size and surface charge. Using a cell counter based method, light transmission aggregometry and scanning electron microscopy, we show that only large cationic dendrimers, but not anionic, neutral or small cationic dendrimers, induce aggregation of human platelets in plasma in vitro. The aggregation caused by large cationic dendrimers was proportional to the number of surface amines. The observed aggregation was not associated with membrane microparticle release, and was insensitive to a variety of chemical and biological inhibitors known to interfere with various pathways of platelet activation. Taken in context with previously reported studies, our data suggest that large cationic PAMAM dendrimers induce platelet aggregation through disruption of membrane integrity.

Molecular heterogeneity analysis of poly(amidoamine) dendrimer-based mono- and multifunctional nanodevices by capillary electrophoresis.

Poly(amidoamine) (PAMAM) dendrimer-based nanodevices are of recent interest in targeted cancer therapy. Characterization of mono- and multifunctional PAMAM-based nanodevices remains a great challenge because of their molecular complexity. In this work, various mono- and multifunctional nanodevices based on PAMAM G5 (generation 5) dendrimer were characterized by UV-Vis spectrometry, (1)H NMR, size exclusion chromatography (SEC), and capillary electrophoresis (CE). CE was extensively utilized to measure the molecular heterogeneity of these PAMAM-based nanodevices. G5-FA (FA denotes folic acid) conjugates (synthesized from amine-terminated G5.NH(2) dendrimer, approach 1) with acetamide and amine termini exhibit bimodal or multi-modal distributions. In contrast, G5-FA and bifunctional G5-FA-MTX (MTX denotes methotrexate) conjugates with hydroxyl termini display a single modal distribution. Multifunctional G5.Ac(n)-FI-FA, G5.Ac(n)-FA-OH-MTX, and G5.Ac(n)-FI-FA-OH-MTX (Ac denotes acetamide; FI denotes fluorescein) nanodevices (synthesized from partially acetylated G5 dendrimer, approach 2) exhibit a monodisperse distribution. It indicates that the molecular distribution of PAMAM conjugates largely depends on the homogeneity of starting materials, the synthetic approaches, and the final functionalization steps. Hydroxylation functionalization of dendrimers masks the dispersity of the final PAMAM nanodevices in both synthetic approaches. The applied CE analysis of mono- and multifunctional PAMAM-based nanodevices provides a powerful tool to evaluate the molecular heterogeneity of complex dendrimer conjugate nanodevices for targeted cancer therapeutics.

Dendrimer-induced leukocyte procoagulant activity depends on particle size and surface charge

AIMSnThrombogenicity associated with the induction of leukocyte procoagulant activity (PCA) is a common complication in sepsis and cancer. Since nanoparticles are increasingly used for drug delivery, their interaction with coagulation systems is an important part of the safety assessment. The purpose of this study was to investigate the effects of nanoparticle physicochemical properties on leukocyte PCA, and to get insight into the mechanism of PCA induction.nnnMATERIALS & METHODSnA total of 12 formulations of polyamidoamine (PAMAM) dendrimers, varying in size and surface charge, were studied in vitro using recalcification time assay.nnnRESULTSnIrrespective of their size, anionic and neutral dendrimers did not induce leukocyte PCA in vitro. Cationic particles induced PCA in a size- and charge-dependent manner. The mechanism of PCA induction was similar to that of doxorubicin. Cationic dendrimers were also found to exacerbate endotoxin-induced PCA.nnnCONCLUSIONnPAMAM dendrimer-induced leukocyte PCA depends on particle size, charge and density of surface groups.

Adverse effects of fullerenes on endothelial cells: Fullerenol C60(OH)24 induced tissue factor and ICAM-1 membrane expression and apoptosis in vitro

We studied the effects of a C60 water suspension at 4 μg/mL (nC60) and the water soluble fullerenol C60(OH)24 at final concentrations of 1–100 μg/mL on human umbilical vein endothelial cells (HUVECs) in culture. We found that a 24 hr treatment of HUVECs with C60(OH)24 at 100 μg/mL significantly increased cell surface expression of ICAM-1(CD54) (67 ± 4% CD54+ cells vs. 19 ± 2 % CD54+ cells in control; p < 0.001). In addition, this treatment induced the expression of tissue factor (CD142) on HUVECs (54 ± 20% CD142+ cells vs 4 ± 2% CD142+ cells in control; p = 0.008) and increased exposure of phosphatidylserine (PS) (29 ± 2% PS+ cells vs. 12 ± 5% PS+ cells in control; p < 0.001). Analysis of cell cycle and DNA fragmentation (TUNEL) showed that both nC60 and C60(OH)24 caused G1 arrest of HUVECs and C60(OH)24 induced significant apoptosis (21 ± 2% TUNEL+ cells at 100 μg/mL of C60(OH)24 vs. 4 ± 2% TUNEL+ cells in control; p < 0.001). We also demonstrated that both nC60 and C60(OH)24 induced a rapid concentration dependent elevation of intracellular calcium [Ca2+]i. This could be inhibited by EGTA, suggesting that the source of [Ca2+]i in fullerene stimulated calcium flux is predominantly from the extracellular environment. In conclusion, fullerenol C60(OH)24 had both pro-inflammatory and pro-apoptotic effects on HUVECs, indicating possible adverse effects of fullerenes on the endothelium.

Sterilization of Silver Nanoparticles Using Standard Gamma Irradiation Procedure Affects Particle Integrity and Biocompatibility

Silver nanoparticles are commonly used in a variety of commercial and medical products. Here we investigate the effects of standard sterilization methods, including heat/steam (autoclave) and gamma-irradiation on the structural integrity and biocompatibility of citrate-stabilized silver nanoparticles with nominal sizes of 20, 40, 60 and 80 nm. Particle size, shape and in vitro biocompatibility were studied pre- and post-sterilization. Sterilization by gamma irradiation at dose levels commonly used in medical device industry (15, 25 and 50 kGy) resulted in dramatic changes in particle size and morphology, as monitored by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Exposing the particles to a chemical producer of hydroxyl radicals (N-hydroxy-2-pyridinethione) allowed us to duplicate the sterilization-based changes in size and morphology, implying a free radical mechanism of action. Compared to untreated controls, we also observed a three- to five-fold increase in tendency of sterilized silver nanoparticles to cause platelet aggregation, a sensitive in vitro indicator of thrombogenicity.

Preclinical Characterization of Engineered Nanoparticles Intended for Cancer Therapeutics

7.

Inhibition of phosphoinositol 3 kinase contributes to nanoparticle-mediated exaggeration of endotoxin-induced leukocyte procoagulant activity

AIMnDisseminated intravascular coagulation is an increasing concern for certain types of engineered nanomaterials. Recent studies have shed some light on the nanoparticle physicochemical properties contributing to this toxicity; however, the mechanisms are poorly understood. Leukocyte procoagulant activity (PCA) is a key factor contributing to the initiation of this toxicity. We have previously reported on the exaggeration of endotoxin-induced PCA by cationic dendrimers. Herein, we report an effort to discern the mechanism.nnnMATERIALS & METHODSnPoly(amidoamine) dendrimers with various sizes and surface functionalities were studied in vitro by the recalcification test, flow cytometry and other relevant assays.nnnRESULTS & CONCLUSIONnCationic dendrimers exaggerated endotoxin-induced PCA, but their anionic or neutral counterparts did not; the cationic charge prompts this phenomenon, but different cationic surface chemistries do not influence it. Cationic dendrimers and endotoxin differentially affect the PCA complex. The inhibition of phosphoinositol 3 kinase by dendrimers contributes to the exaggeration of the endotoxin-induced PCA.