Santimukul Santra

Oxidase activity of polymeric coated cerium oxide nanoparticles

Inorganic enzyme? Ceria nanoparticles exhibit unique oxidase-like activity at acidic pH values. These redox catalysts can be used in immunoassays (ELISA) when modified with targeting ligands (see picture; light blue and yellow structures are nanoparticles with attached ligands). This modification allows both for binding and for detection by the catalytic oxidation of sensitive colorimetric dyes (e.g. TMB).

Drug/Dye‐Loaded, Multifunctional Iron Oxide Nanoparticles for Combined Targeted Cancer Therapy and Dual Optical/Magnetic Resonance Imaging

A biocompatible, multimodal, and theranostic functional iron oxide nanoparticle is synthesized using a novel water-based method and exerts excellent properties for targeted cancer therapy, and optical and magnetic resonance imaging. For the first time, a facile, modified solvent diffusion method is used for the co-encapsulation of both an anticancer drug and near-infrared dyes. The resulting folate-derivatized theranostics nanoparticles could allow for targeted optical/magnetic resonance imaging and targeted killing of folate-expressing cancer cells.

Role of nanoparticle valency in the nondestructive magnetic-relaxation-mediated detection and magnetic isolation of cells in complex media.

Nanoparticle-based diagnostics typically involve the conjugation of targeting ligands to the nanoparticle to create a sensitive and specific nanosensor that can bind and detect the presence of a target, such as a bacterium, cancer cell, protein, or DNA sequence. Studies that address the effect of multivalency on the binding and detection pattern of these nanosensors, particularly on magnetic relaxation nanosensors that sense the presence of a target in a dose-dependent manner by changes in the water relaxation times (DeltaT2), are scarce. Herein, we study the effect of multivalency on the detection profile of cancer cells and bacteria in complex media, such as blood and milk. In these studies, we conjugated folic acid at two different densities (low-folate and high-folate) on polyacrylic-acid-coated iron oxide nanoparticles and studied the interaction of these magnetic nanosensors with cancer cells expressing the folate receptor. Results showed that the multivalent high-folate magnetic relaxation nanosensor performed better than its low folate counterpart, achieving single cancer cell detection in blood samples within 15 min. Similar results were also observed when a high molecular weight anti-folate antibody (MW 150 kDa) was used instead of the low molecular weight folic acid ligand (MW 441.4 kDa), although better results in terms of sensitivity, dynamic range, and speed of detection were obtained when the folate ligand was used. Studies using bacteria in milk suspensions corroborated the results observed with cancer cells. Taken together, these studies demonstrate that nanoparticle multivalency plays a key role in the interaction of the nanoparticle with the cellular target and modulate the behavior and sensitivity of the assay. Furthermore, as detection with magnetic relaxation nanosensors is a nondestructive technique, magnetic isolation and further characterization of the cancer cells is possible.

Cytochrome c Encapsulating Theranostic Nanoparticles: A Novel Bifunctional System for Targeted Delivery of Therapeutic Membrane-Impermeable Proteins to Tumors and Imaging of Cancer Therapy

The effective administration of therapeutic proteins has received increased attention for the treatment of various diseases. Encapsulation of these proteins in various matrices, as a method of protein structure and function preservation, is a widely used approach that results in maintenance of the proteins function. However, targeted delivery and tracking of encapsulated therapeutic proteins to the affected cells is still a challenge. In an effort to advance the targeted delivery of a functional apoptosis-initiating protein (cytochrome c) to cancer cells, we formulated theranostic polymeric nanoparticles for the simultaneous encapsulation of cytochrome c and a near-infrared dye to folate-expressing cancer cells. The polymeric nanoparticles were prepared using a novel water-soluble hyperbranched polyhydroxyl polymer that allows for dual encapsulation of a hydrophilic protein and an amphiphilic fluorescent dye. Our protein therapeutic cargo is the endogenous protein cytochrome c, which upon cytoplasmic release, initiates an apoptotic response leading to programmed cell death. Results indicate that encapsulation of cytochrome c within the nanoparticles cavities preserved the proteins enzymatic activity. The potential therapeutic property of these nanoparticles was demonstrated by the induction of apoptosis upon intracellular delivery. Furthermore, targeted delivery of cytochrome c to folate-receptor-positive cancer cells was achieved via conjugation of folic acid to the nanoparticles surface, whereas the nanoparticles theranostic properties were conferred via the coencapsulation of cytochrome c and a fluorescent dye. Considering that these theranostic nanoparticles can carry an endogenous cellular apoptotic initiator (cytochrome c) and a fluorescent tag (ICG) commonly used in the clinic, their use and potential translation into the clinic is anticipated, facilitating the monitoring of tumor regression.

Gadolinium-Encapsulating Iron Oxide Nanoprobe as Activatable NMR/MRI Contrast Agent

Herein we report a novel gadolinium-encapsulating iron oxide nanoparticle-based activatable NMR/MRI nanoprobe. In our design, Gd-DTPA is encapsulated within the poly(acrylic acid) (PAA) polymer coating of a superparamagnetic iron oxide nanoparticle (IO-PAA), yielding a composite magnetic nanoprobe (IO-PAA-Gd-DTPA) with quenched longitudinal spin-lattice magnetic relaxation (T(1)). Upon release of the Gd-DTPA complex from the nanoprobes polymeric coating in acidic media, an increase in the T(1) relaxation rate (1/T(1)) of the composite magnetic nanoprobe was observed, indicating a dequenching of the nanoprobe with a corresponding increase in the T(1)-weighted MRI signal. When a folate-conjugated nanoprobe was incubated in HeLa cells, a cancer cell line overexpressing folate receptors, an increase in the 1/T(1) signal was observed. This result suggests that, upon receptor-mediated internalization, the composite magnetic nanoprobe degraded within the cells lysosome acidic (pH 5.0) environment, resulting in an intracellular release of Gd-DTPA complex with subsequent T(1) activation. In addition, when an anticancer drug (Taxol) was coencapsulated with the Gd-DTPA within the folate receptor targeting composite magnetic nanoprobe, the T(1) activation of the probe coincided with the rate of drug release and corresponding cytotoxic effect in cell culture studies. Taken together, these results suggest that our activatable T(1) nanoagent could be of great importance for the detection of acidic tumors and assessment of drug targeting and release by MRI.

Aliphatic Hyperbranched Polyester: A New Building Block in the Construction of Multifunctional Nanoparticles and Nanocomposites

Herein we report the design and synthesis of multifunctional hyperbranched polyester-based nanoparticles and nanocomposites with properties ranging from magnetic, fluorescence, antioxidant and X-ray contrast. The fabrication of these nanostructures was achieved using a novel aliphatic and biodegradable hyperbranched polyester (HBPE) synthesized from readily available diethyl malonate. The polymers globular structure with functional surface carboxylic groups and hydrophobic cavities residing in the polymers interior allows for the formation of multifunctional polymeric nanoparticles, which are able to encapsulate a diversity of hydrophobic cargos. Via simple surface chemistry modifications, the surface carboxylic acid groups were modified to yield nanoparticles with a variety of surface functionalizations, such as amino, azide and propargyl groups, which mediated the conjugation of small molecules. This capability achieved the engineering of the HBPE nanoparticle surface for specific cell internalization studies and the formation of nanoparticle assemblies for the creation of novel nanocomposites that retained, and in some cases enhanced, the properties of the parental nanoparticle building blocks. Considering these results, the HBPE polymer, nanoparticles and composites should be ideal for biomedical, pharmaceutical, nanophotonics applications.

Environment-responsive nanophores for therapy and treatment monitoring via molecular MRI quenching

The effective delivery of therapeutics to disease sites significantly contributes to drug efficacy, toxicity and clearance. Here we demonstrate that clinically approved iron oxide nanoparticles (Ferumoxytol) can be utilized to carry one or multiple drugs. These so called ‘nanophores’ retain their cargo within their polymeric coating through weak electrostatic interactions and release it in slightly acidic conditions (pH 6.8 and below). The loading of drugs increases the nanophores’ transverse T2 and longitudinal T1 NMR proton relaxation times, which is proportional to amount of carried cargo. Chemotherapy with translational nanophores is more effective than the free drug in vitro and in vivo, without subjecting the drugs or the carrier nanoparticle to any chemical modification. Evaluation of cargo incorporation and payload levels in vitro and in vivo can be assessed via benchtop magnetic relaxometers, common NMR instruments or MRI scanners.

Rapid and Sensitive Detection of an Intracellular Pathogen in Human Peripheral Leukocytes with Hybridizing Magnetic Relaxation Nanosensors

Bacterial infections are still a major global healthcare problem. The quick and sensitive detection of pathogens responsible for these infections would facilitate correct diagnosis of the disease and expedite treatment. Of major importance are intracellular slow-growing pathogens that reside within peripheral leukocytes, evading recognition by the immune system and detection by traditional culture methods. Herein, we report the use of hybridizing magnetic nanosensors (hMRS) for the detection of an intracellular pathogen, Mycobacterium avium spp. paratuberculosis (MAP). The hMRS are designed to bind to a unique genomic sequence found in the MAP genome, causing significant changes in the sample’s magnetic resonance signal. Clinically relevant samples, including tissue and blood, were screened with hMRS and results were compared with traditional PCR analysis. Within less than an hour, the hMRS identified MAP-positive samples in a library of laboratory cultures, clinical isolates, blood and homogenized tissues. Comparison of the hMRS with culture methods in terms of prediction of disease state revealed that the hMRS outperformed established culture methods, while being significantly faster (1 hour vs 12 weeks). Additionally, using a single instrument and one nanoparticle preparation we were able to detect the intracellular bacterial target in clinical samples at the genomic and epitope levels. Overall, since the nanoparticles are robust in diverse environmental settings and substantially more affordable than PCR enzymes, the potential clinical and field-based use of hMRS in the multiplexed identification of microbial pathogens and other disease-related biomarkers via a single, deployable instrument in clinical and complex environmental samples is foreseen.

Rational development of a cytotoxic peptide to trigger cell death

Defects in the apoptotic machinery can contribute to tumor formation and resistance to treatment, creating a need to identify new agents that kill cancer cells by alternative mechanisms. To this end, we examined the cytotoxic properties of a novel peptide, CT20p, derived from the C-terminal, alpha-9 helix of Bax, an amphipathic domain with putative membrane binding properties. Like many antimicrobial peptides, CT20p contains clusters of hydrophobic and cationic residues that could enable the peptide to associate with lipid membranes. CT20p caused the release of calcein from mitochondrial-like lipid vesicles without disrupting vesicle integrity and, when expressed as a fusion protein in cells, localized to mitochondria. The amphipathic nature of CT20p allowed it to be encapsulated in polymeric nanoparticles (NPs) that have the capacity to harbor targeting molecules, dyes or drugs. The resulting CT20p-NPs proved an effective killer, in vitro, of colon and breast cancer cells, and in vivo, using a murine breast cancer tumor model. By introducing CT20p to Bax deficient cells, we demonstrated that the peptides lethal activity was independent of endogenous Bax. CT20p also caused an increase in the mitochondrial membrane potential that was followed by plasma membrane rupture and cell death, without the characteristic membrane asymmetry associated with apoptosis. We determined that cell death triggered by the CT20p-NPs was minimally dependent on effector caspases and resistant to Bcl-2 overexpression, suggesting that it acts independently of the intrinsic apoptotic death pathway. Furthermore, use of CT20p with the apoptosis-inducing drug, cisplatin, resulted in additive toxicity. These results reveal the novel features of CT20p that allow nanoparticle-mediated delivery to tumors and the potential application in combination therapies to activate multiple death pathways in cancer cells.

Selective N-alkylation of β-alanine facilitates the synthesis of a poly(amino acid)-based theranostic nanoagent.

The development of functional amino acid-based polymeric materials is emerging as a platform to create biodegradable and nontoxic nanomaterials for medical and biotechnology applications. In particular, facile synthetic routes for these polymers and their corresponding polymeric nanomaterials would have a positive impact in the development of novel biomaterials and nanoparticles. However, progress has been hampered by the need to use complex protection-deprotection methods and toxic phase transfer catalysts. In this study, we report a facile, single-step approach for the synthesis of an N-alkylated amino acid as an AB-type functional monomer to generate a novel pseudo-poly(amino acid), without using the laborious multistep, protection-deprotection methods. This synthetic strategy is reproducible, easy to scale up, and does not produce toxic byproducts. In addition, the synthesized amino acid-based polymer is different from conventional linear polymers as the butyl pendants enhance its solubility in common organic solvents and facilitate the creation of hydrophobic nanocavities for the effective encapsulation of hydrophobic cargos upon nanoparticle formation. Within the nanoparticles, we have encapsulated a hydrophobic DiI dye and a therapeutic drug, Taxol. In addition, we have conjugated folic acid as a folate receptor-targeting ligand for the targeted delivery of the nanoparticles to cancer cells expressing the folate receptor. Cell cytotoxicity studies confirm the low toxicity of the polymeric nanoparticles, and drug-release experiments with the Taxol-encapsulated nanoparticles only exhibit cytotoxicity upon internalization into cancer cells expressing the folate receptor. Taken together, these results suggested that our synthetic strategy can be useful for the one-step synthesis of amino acid-based small molecules, biopolymers, and theranostic polymeric nanoagents for the targeted detection and treatment of cancer.