Vahid Khalilzad-Sharghi

PSMA targeted docetaxel-loaded superparamagnetic iron oxide nanoparticles for prostate cancer.

Docetaxel (Dtxl) is currently the most common therapeutic option for prostate cancer (PC). However, adverse side effects and problems associated with chemo-resistance limit its therapeutic outcome in clinical settings. A targeted nanoparticle system to improve its delivery to and activity at the tumor site could be an attractive strategy for PC therapy. Therefore, the objective of this study was to develop and determine the anti-cancer efficacy of a novel docetaxel loaded, prostate specific membrane antigen (PSMA) targeted superparamagnetic iron oxide nanoparticle (SPION) (J591-SPION-Dtxl) formulation for PC therapy. Our results showed the SPION-Dtxl formulation exhibits an optimal particle size and zeta potential, which can efficiently be internalized in PC cells. SPION-Dtxl exhibited potent anti-cancer efficacy via induction of the expression of apoptosis associated proteins, downregulation of anti-apoptotic proteins, and inhibition of chemo-resistance associated protein in PC cell lines. J591-SPION-Dtxl exhibited a profound uptake in C4-2 (PSMA(+)) cells compared to PC-3 (PSMA(-)) cells. A similar targeting potential was observed in ex-vivo studies in C4-2 tumors but not in PC-3 tumors, suggesting its tumor specific targeting. Overall, this study suggests that a PSMA antibody functionalized SPION-Dtxl formulation can be highly useful for targeted PC therapy.

Magnetic resonance elastography methodology for the evaluation of tissue engineered construct growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development. The method proposed in the following protocol uses microscopic magnetic resonance elastography (μMRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner. Recently, μMRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map. The resulting measurements at each voxel have been shown to strongly correlate (R(2)>0.9914) with data collected using dynamic mechanical analysis. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

Localization of CD8 T cell epitope within cardiac myosin heavy chain-α334-352 that induces autoimmune myocarditis in A/J mice

BACKGROUND Cardiac myosin heavy chain-α (Myhc), an intracellular protein expressed in the cardiomyocytes, has been identified as a major autoantigen in cardiac autoimmunity. In our studies with Myhc334-352-induced experimental autoimmune myocarditis in A/J mice (H-2a), we discovered that Myhc334-352, supposedly a CD4 T cell epitope, also induced antigen-specific CD8 T cells that transfer disease to naive animals. METHODS AND RESULTS In our efforts to identify the CD8 T cell determinants, we localized Myhc338-348 within the full length-Myhc334-352, leading to four key findings. (1) By acting as a dual epitope, Myhc338-348 induces both CD4 and CD8 T cell responses. (2) In a major histocompatibility complex (MHC) class I-stabilization assay, Myhc338-348 was found to bind H-2Dd-but not H-2Kk or H-2Ld-alleles. (3) The CD8 T cell response induced by Myhc338-348 was antigen-specific, as evaluated by MHC class I/H-2Dd dextramer staining. The antigen-sensitized T cells predominantly produced interferon-γ, the critical cytokine of effector cytotoxic T lymphocytes. (4) Myhc338-348 was found to induce myocarditis in immunized animals as determined by histology and magnetic resonance microscopy imaging. CONCLUSIONS Our data provide new insights as to how different immune cells can recognize the same antigen and inflict damage through different mechanisms.

Noninvasive assessment of cardiac abnormalities in experimental autoimmune myocarditis by magnetic resonance microscopy imaging in the mouse.

Myocarditis is an inflammation of the myocardium, but only -10% of those affected show clinical manifestations of the disease. To study the immune events of myocardial injuries, various mouse models of myocarditis have been widely used. This study involved experimental autoimmune myocarditis (EAM) induced with cardiac myosin heavy chain (Myhc)-α 334-352 in A/J mice; the affected animals develop lymphocytic myocarditis but with no apparent clinical signs. In this model, the utility of magnetic resonance microscopy (MRM) as a non-invasive modality to determine the cardiac structural and functional changes in animals immunized with Myhc-α 334-352 is shown. EAM and healthy mice were imaged using a 9.4 T (400 MHz) 89 mm vertical core bore scanner equipped with a 4 cm millipede radio-frequency imaging probe and 100 G/cm triple axis gradients. Cardiac images were acquired from anesthetized animals using a gradient-echo-based cine pulse sequence, and the animals were monitored by respiration and pulse oximetry. The analysis revealed an increase in the thickness of the ventricular wall in EAM mice, with a corresponding decrease in the interior diameter of ventricles, when compared with healthy mice. The data suggest that morphological and functional changes in the inflamed hearts can be non-invasively monitored by MRM in live animals. In conclusion, MRM offers an advantage of assessing the progression and regression of myocardial injuries in diseases caused by infectious agents, as well as response to therapies.

Identification of an Epitope from Adenine Nucleotide Translocator 1 That Induces Inflammation in Heart in A/J Mice

Heart failure, a leading cause of death in humans, can emanate from myocarditis. Although most individuals with myocarditis recover spontaneously, some develop chronic dilated cardiomyopathy. Myocarditis may result from both infectious and noninfectious causes, including autoimmune responses to cardiac antigens. In support of this notion, intracellular cardiac antigens, like cardiac myosin heavy chain-α, cardiac troponin-I, and adenine nucleotide translocator 1 (ANT1), have been identified as autoantigens in cardiac autoimmunity. Herein, we demonstrate that ANT1 can induce autoimmune myocarditis in A/J mice by generating autoreactive T cells. We show that ANT1 encompasses multiple immunodominant epitopes (namely, ANT1 21-40, ANT1 31-50, ANT1 171-190, and ANT1 181-200). Although all four peptides induce comparable T-cell responses, only ANT1 21-40 was found to be a major myocarditogenic epitope in immunized animals. The myocarditis-inducing ability of ANT1 21-40 was associated with the generation of T cells producing predominantly IL-17A, and the antigen-sensitized T cells could transfer the disease to naïve recipients. These data indicate that cardiac mitochondrial proteins can be target autoantigens in myocarditis, supporting the notion that the antigens released as a result of primary damage may contribute to the persistence of chronic inflammation through autoimmunity.

MR elastography for evaluating regeneration of tissue-engineered cartilage in an ectopic mouse model.

The purpose of the present study was to apply noninvasive methods for monitoring regeneration and mechanical properties of tissue‐engineered cartilage in vivo at different growth stages using MR elastography (MRE).

Theranostic fluorescent silica encapsulated magnetic nanoassemblies for in vitro MRI imaging and hyperthermia

This article reports the synthesis of manganese ferrite nano-assemblies (MNAs) encapsulated with fluorescent silica shell and demonstrates their applicability for magnetic hyperthermia, optical and T2 contrast MRI imaging with HeLa cancer cells. The MNAs were encapsulated by a double layer of silica shell through a two-step sol–gel process. The inner silica shell contains rhodamine-B isothiocyanate (RITC) dye, whereas the outer silica layer is without RITC-dye, helps to prevent photo-bleaching and increase photo-luminance. MNAs@Dye–SiO2@SiO2 exhibited a high magnetization of 90.43 emu g−1 with a remarkably high r2 value of 598 ± 2 mM−1 s−1 (Mn + Fe). The cellular uptake of MNAs@Dye–SiO2@SiO2 was observed by the presence of fluorescent red granulated spots in the cytoplasm of HeLa cells, confirming its efficacy for optical imaging. High transverse relaxivities r2 (darkening effect) were observed in HeLa cells incubated with MNAs@Dye–SiO2@SiO2 in comparison to HeLa cells without particles. An 80–85% cell death was achieved on induction of magnetic hyperthermia with HeLa cells at the lowest Hf factor value (3.3 × 109 A m−1 s−1). Our results show MNAs@Dye–SiO2@SiO2 as a novel multifunctional theranostic nanoprobe, which can realize its applicability for diagnostic and real time monitoring the efficacy of ongoing cancer therapy.

Abstract 4459: Characterization of a novel magnetic nanoparticles formulation for cancer therapeutic applications

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA We have successfully engineered a magnetic nanoparticles (MAG-NPs) formulation using a multi-layer approach which can be used for drug/gene/biomolecule delivery, hyperthermia, and magnetic resonance imaging (MRI) applications in cancer therapeutics. Overcoming nanoparticles clearance by the immune system remains a major challenge. This formulation is designed to provide an additional surface layer as molecular “authentication” that the body does not recognize as foreign material. The interaction between the surface of nanoparticles and plasma proteins leads to nanoparticle-protein complex which determines the rational design of clinically useful formulations for cancer therapeutics. This formulation was very effective for inhibiting tumorigenic features in in vitro and in vivo cancer models. Therefore, we further studied our MAG-NPs formulation for biologically and clinically relevant characteristic features for its clinical translation. For this, we have examined the change in particle size, zeta potential, human serum protein adsorption, hemotoxicity, uptake, targeting cancer cells, and magnetic resonance imaging of the MAG-NPs formulation. No significant change was observed in particle size and zeta potential of nanoformulation with the incubation of 0.5 to 50 wt/wt% human serum. With increase of serum concentration and particle concentration there was a clear indication that apart from serum albumin and transferrin; apolipoprotein E (Apo-E, a surrogate marker for improved circulation of nanoparticles and enhanced crossing of the blood-brain barrier) also adsorbed on the surface of nanoparticles (determined by Western blot analysis). No significant primary or secondary structural alterations were observed in serum proteins through circular dichroism study. Hemolysis assay demonstrated almost no hemolysis at the tested concentrations (up to 1 mg/mL) for nanoparticles compared to the positive control (sodium dodecyl sulphate). Particle internalization and targeting cancer cells were observed in dose dependent manner and were not influenced by protein architecture on surface of nanoparticles. Additionally, this formulation has exhibited identical T1 relaxation times (∼1.1-1.6 s) and T2 relaxation times (∼ 13-14 ms) which suggests that our nanoparticles retained MRI properties even after serum proteins adsorption and drug/biomolecular encapsulation. Based on all these superior clinical characteristics, this MAG-NPs formulation can effectively be used for the delivery of drug/siRNA/miRNAs for cancer therapy, diagnosis, and imaging. Citation Format: Murali M. Yallapu, Neeraj Chauhan, Shadi F. Othman, Vahid Khalilzad-Sharghi, Meena Jaggi, Subhash C. Chauhan. Characterization of a novel magnetic nanoparticles formulation for cancer therapeutic applications. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4459. doi:10.1158/1538-7445.AM2014-4459

Multi‐modal imaging for assessment of tissue‐engineered bone in a critical‐sized calvarial defect mouse model

Tissue‐engineered bone (TEB) analysis in vivo relies heavily on tissue histological and end‐point evaluations requiring the sacrifice of animals at specific time points. Due to differences in animal response to implanted tissues, the conventional analytical methods to evaluate TEB can introduce data inconsistencies. Additionally, the conventional methods increase the number of animals required to provide an acceptable statistical power for hypothesis testing. Alternatively, our non‐invasive optical imaging allows for the longitudinal analysis of regenerating tissue, where each animal acts as its own control, thus reducing overall animal numbers. In our 6 month feasibility study, TEB, consisting of a silk protein scaffold with or without differentiated mesenchymal stem cells, was implanted in a critical‐sized calvarial defect mouse model. Osteogenesis of the TEB was monitored through signal variation, using magnetic resonance imaging (MRI) and near‐infrared (NIR) optical imaging with IRDye® 800CW BoneTagTM (800CW BT, a bone‐specific marker used to label osteogenically differentiated mesenchymal stem cells and mineralization). Histological endpoint measurements and computed tomography (CT) were used to confirm imaging findings. Anatomical MRI revealed decreased signal intensity, indicating mineralization, in the TEB compared to the control (i.e. silk scaffold only) at various growth stages. NIR optical imaging results demonstrated a signal intensity increase of the TEB compared to control. Interpretation of the imaging results were confirmed by histological analysis. Specifically, haematoxylin and eosin staining revealing de novo bone in TEB showed that 80% of the defect was covered by TEB, while only 40% was covered for the control. Taken together, these results demonstrate the potential of multi‐modal non‐invasive imaging to visualize and quantify TEB for the assessment of regenerative medicine strategies. Copyright

Epitope Mapping of SERCA2a Identifies an Antigenic Determinant That Induces Mainly Atrial Myocarditis in A/J Mice

Sarcoplasmic/endoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA)2a, a critical regulator of calcium homeostasis, is known to be decreased in heart failure. Patients with myocarditis or dilated cardiomyopathy develop autoantibodies to SERCA2a suggesting that they may have pathogenetic significance. In this report, we describe epitope mapping analysis of SERCA2a in A/J mice that leads us to make five observations: 1) SERCA2a contains multiple T cell epitopes that induce varying degrees of myocarditis. One epitope, SERCA2a 971–990, induces widespread atrial inflammation without affecting noncardiac tissues; the cardiac abnormalities could be noninvasively captured by echocardiography, electrocardiography, and magnetic resonance microscopy imaging. 2) SERCA2a 971–990-induced disease was associated with the induction of CD4 T cell responses and the epitope preferentially binds MHC class II/IAk rather than IEk. By creating IAk/and IEk/SERCA2a 971–990 dextramers, the T cell responses were determined by flow cytometry to be Ag specific. 3) SERCA2a 971–990-sensitized T cells produce both Th1 and Th17 cytokines. 4) Animals immunized with SERCA2a 971–990 showed Ag-specific Abs with enhanced production of IgG2a and IgG2b isotypes, suggesting that SERCA2a 971–990 can potentially act as a common epitope for both T cells and B cells. 5) Finally, SERCA2a 971–990-sensitized T cells were able to transfer disease to naive recipients. Together, these data indicate that SERCA2a is a critical autoantigen in the mediation of atrial inflammation in mice and that our model may be helpful to study the inflammatory events that underlie the development of conditions such as atrial fibrillation in humans.