Gold and iron oxide hybrid nanoparticles (HNPs) synthesized by the thermal decomposition technique are bio-functionalized with a single chain antibody, scFv, that binds to the A33 antigen present on colorectal cancer cells. The HNP-scFv conjugates are stable in aqueous solution with a magnetization value of 44 emu g(-1) and exhibit strong optical absorbance at 800 nm. Here we test this material in targeting, imaging and selective thermal killing of colorectal cancer cells. Cellular uptake studies showed that A33-expressing cells take up the A33scFv-conjugated HNPs at a rate five times higher than cells that do not express the A33 antigen. Laser irradiation studies showed that approximately 53% of the A33-expressing cells exposed to targeted HNPs are killed after a six-minute laser treatment at 5.1 W cm(-2) using a 808 nm continuous wave laser diode while < 5% of A33-nonexpressing cells are killed. At a higher intensity, 31.5 W cm(-2), the thermal destruction increases to 99 and 40% for A33-expressing cells and A33 nonexpressing cells, respectively, after 6 min exposure. Flow cytometric analyses of the laser-irradiated A33 antigen-expressing cells show apoptosis-related cell death to be the primary mode of cell death at 5.1 W cm(-2), with increasing necrosis-related cell death at higher laser power. These results suggest that this new class of bio-conjugated hybrid nanoparticles can potentially serve as an effective antigen-targeted photothermal therapeutic agent for cancer treatment as well as a probe for magnetic resonance-based imaging.

Understanding interactions occurring at the interface between nanoparticles and biological components is an urgent challenge in nanomedicine due to their effect on the biological fate of nanoparticles. After the systemic injection of nanoparticles, a protein corona constructed by blood components surrounds the carrier's surface and modulates its pharmacokinetics and biodistribution. Biomimicry-based approaches in nanotechnology attempt to imitate what happens in nature in order to transfer specific natural functionalities to synthetic nanoparticles. Several biomimetic formulations have been developed, showing superior in vivo features as a result of their cell-like identity. We have recently designed biomimetic liposomes, called leukosomes, which recapitulate the ability of leukocytes to target inflamed endothelium and escape clearance by the immune system. To gain insight into the properties of leukosomes, we decided to investigate their protein corona in vivo. So far, most information about the protein corona has been obtained using in vitro experiments, which have been shown to minimally reproduce in vivo phenomena. Here we directly show a time-dependent quantitative and qualitative analysis of the protein corona adsorbed in vivo on leukosomes and control liposomes. We observed that leukosomes absorb fewer proteins than liposomes, and we identified a group of proteins specifically adsorbed on leukosomes. Moreover, we hypothesize that the presence of macrophage receptors on leukosomes' surface neutralizes their protein corona-meditated uptake by immune cells. This work unveils the protein corona of a biomimetic carrier and is one of the few studies on the corona performed in vivo.

Effective delivery holds the key to successful in vivo application of therapeutic small interfering RNA (siRNA). In this work, we have developed a universal siRNA carrier consisting of a mesoporous silica nanoparticle (MSNP) functionalized with cyclodextrin-grafted polyethylenimine (CP). CP provides positive charge for loading of siRNA through electrostatic interaction and enables effective endosomal escape of siRNA. Using intravital microscopy we were able to monitor tumor enrichment of CP-MSNP/siRNA particles in live mice bearing orthotopic MDA-MB-231 xenograft tumors. CP-MSNP delivery of siRNA targeting the M2 isoform of the glycolytic enzyme pyruvate kinase (PKM2) resulted in effective knockdown of gene expression in vitro and in vivo. Suppression of PKM2 led to inhibition of tumor cell growth, invasion, and migration.

Understanding the mechanism of radioresistance could help develop strategies to improve therapeutic response of patients with PDAC. The gene is frequently mutated in pancreatic cancer. In this study, we investigated the role of deficiency in pancreatic cancer cells' response to radiotherapy. We downregulated SMAD4 expression with siRNA or shRNA and overexpressed SMAD4 in mutant pancreatic cancer cells followed by clonogenic survival assay to evaluate their effects on cell radioresistance. To study the mechanism of radioresistance, the effects of loss on reactive oxygen species (ROS) and autophagy were determined by flow cytometry and immunoblot analysis, respectively. Furthermore, we measured radioresistance by clonogenic survival assay after treatment with autophagy inhibitor (Chloroquine) and ROS inhibitor (N-acetyl-l-cysteine) in -depleted pancreatic cancer cells. Finally, the effects of on radioresistance were also confirmed in an orthotopic tumor model derived from -depleted Panc-1 cells.-depleted pancreatic cancer cells were more resistant to radiotherapy based on clonogenic survival assay. Overexpression of wild-type SMAD4 in -mutant cells rescued their radiosensitivity. Radioresistance mediated by depletion was associated with persistently higher levels of ROS and radiation-induced autophagy. Finally, depletion induced radioresistance in Panc-1-derived orthotopic tumor model ( = 0.038). More interestingly, we observed that the protein level of SMAD4 is inversely correlated with autophagy in orthotopic tumor tissue samples. Our results demonstrate that defective is responsible for radioresistance in pancreatic cancer through induction of ROS and increased level of radiation-induced autophagy..

Activation of the vascular endothelium is characterized by increased expression of vascular adhesion molecules and chemokines. This activation occurs early in the progression of several diseases and triggers the recruitment of leukocytes. Inspired by the tropism of leukocytes, we investigated leukocyte-based biomimetic nanoparticles (i.e., leukosomes) as a novel theranostic platform for inflammatory diseases.Methods: Leukosomes were assembled by combining phospholipids and membrane proteins from leukocytes. For imaging applications, phospholipids modified with rhodamine and gadolinium were used. Leukosomes incubated with antibodies blocking lymphocyte function-associated antigen 1 (LFA-1) and CD45 were administered to explore their roles in targeting inflammation. In addition, relaxometric assessment of NPs was evaluated.Results: Liposomes and leukosomes were both spherical in shape with sizes ranging from 140-170 nm. Both NPs successfully integrated 8 and 13 µg of rhodamine and gadolinium, respectively, and demonstrated less than 4% variation in physicochemical features. Leukosomes demonstrated a 16-fold increase in breast tumor accumulation relative to liposomes. Furthermore, quantification of leukosomes in tumor vessels demonstrated a 4.5-fold increase in vessel lumens and a 14-fold increase in vessel walls. Investigating the targeting mechanism of action revealed that blockage of LFA-1 on leukosomes resulted in a 95% decrease in tumor accumulation. Whereas blockage of CD45 yielded a 60% decrease in targeting and significant increases in liver and spleen accumulation. In addition, when administered in mice with atherosclerotic plaques, leukosomes exhibited a 4-fold increase in the targeting of inflammatory vascular lesions. Lastly, relaxometric assessment of NPs demonstrated that the incorporation of membrane proteins into leukosomes did not impact the r1 and r2 relaxivities of the NPs, demonstrating 6 and 30 mM-1s-1, respectively.Conclusion: Our study demonstrates the ability of leukosomes to target activated vasculature and exhibit superior accumulation in tumors and vascular lesions. The versatility of the phospholipid backbone within leukosomes permits the incorporation of various contrast agents. Furthermore, leukosomes can potentially be loaded with therapeutics possessing diverse physical properties and thus warrant further investigation toward the development of powerful theranostic agents.

Genetically-modified T cells expressing chimeric antigen receptors (CAR) exert anti-tumor effect by identifying tumor-associated antigen (TAA), independent of major histocompatibility complex. For maximal efficacy and safety of adoptively transferred cells, imaging their biodistribution is critical. This will determine if cells home to the tumor and assist in moderating cell dose. Here, T cells are modified to express CAR. An efficient, non-toxic process with potential for cGMP compliance is developed for loading high cell number with multi-modal (PET-MRI) contrast agents (Super Paramagnetic Iron Oxide Nanoparticles – Copper-64; SPION-64Cu). This can now be potentially used for 64Cu-based whole-body PET to detect T cell accumulation region with high-sensitivity, followed by SPION-based MRI of these regions for high-resolution anatomically correlated images of T cells. CD19-specific-CAR+SPIONpos T cells effectively target in vitro CD19+ lymphoma.

A key step in particle-based drug delivery throughmicrocirculation is particlemigration from blood flow to vesselwalls, also known as “margination”,which promotes particle contact and adhesion to the vesselwall. Margination and adhesion should be independently addressed as two distinct phenomena, considering that the former is a fundamental prerequisite to achieve particle adhesion and subsequent extravasation. Although margination has beenmodeled by numerical simulations and investigated inmodel systems in vitro, experimental studies including red blood cells (RBCs) are lacking. Here, we evaluate the effect of RBCs on margination through microfluidic studies in vitro and by intravital microscopy in vivo.We showthatmargination,which is almost absent when particles are suspended in a cell-free medium, is drastically enhanced by RBCs. This effect is size- and shape-dependent, larger spherical/discoid particles being more effectively marginated both in vitro and in vivo. Our findings can be explained by the collision of particles with RBCs that induces the drifting of the particles towards the vessel walls where they become trapped in the cell-free layer. These results are relevant for the design of drug delivery strategies based on systemically administered carriers.

The abnormal tumor vasculature presents a major challenge to the adequate delivery of chemotherapeutics, often limiting efficacy. We developed a nanoparticle-based technique to deliver localized mild hyperthermia (MHT) used to transiently alter tumor vascular transport properties and enhance transport of macromolecules into tumor interstitium. The strategy involved administering and localizing accumulation of stealth gold nanorods (GNRs, 103 μg of GNRs/g of tumor), and irradiating tumor with a low-photon laser flux (1 W/cm2) to generate MHT. The treatment increased vascular permeability within 24 h after treatment, allowing enhanced transport of macromolecules up to 54 nm in size. A mathematical model is used to describe changes in tumor mass transport properties where the rate of macromolecular exchange between interstitial and vascular region (R) and maximum dye enhancement (Ymax) of 23-nm dextran dye are analytically solved. During enhanced permeability, R increased by 200% while Ymax increased by 30% relative to untreated group in pancreatic CAPAN-1 tumors. MHT treatment also enhanced transport of larger dextran dye (54 nm) as assessed intravital microscopy, without causing occlusive cellular damage. Enhanced vascular transport was prolonged for up to 24 h after treatment, but reversible with transport parameters returning to basal levels after 36 h. This study indicates that localized mild hyperthermia treatment open a transient time-window with which to enable and augment macromolecule transport and potentially improve therapeutic efficacy.