Suraj Dixit

Transferrin receptor-targeted theranostic gold nanoparticles for photosensitizer delivery in brain tumors

Therapeutic drug delivery across the blood-brain barrier (BBB) is not only inefficient, but also nonspecific to brain stroma. These are major limitations in the effective treatment of brain cancer. Transferrin peptide (Tfpep) targeted gold nanoparticles (Tfpep-Au NPs) loaded with the photodynamic pro-drug, Pc 4, have been designed and compared with untargeted Au NPs for delivery of the photosensitizer to brain cancer cell lines. In vitro studies of human glioma cancer lines (LN229 and U87) overexpressing the transferrin receptor (TfR) show a significant increase in cellular uptake for targeted conjugates as compared to untargeted particles. Pc 4 delivered from Tfpep-Au NPs clusters within vesicles after targeting with the Tfpep. Pc 4 continues to accumulate over a 4 hour period. Our work suggests that TfR-targeted Au NPs may have important therapeutic implications for delivering brain tumor therapies and/or providing a platform for noninvasive imaging.

Dual Receptor-Targeted Theranostic Nanoparticles for Localized Delivery and Activation of Photodynamic Therapy Drug in Glioblastomas

Targeting gold nanoparticles (AuNPs) with two or more receptor binding peptides has been proposed to address intratumoral heterogeneity of glioblastomas that overexpress multiple cell surface receptors to ultimately improve therapeutic efficacy. AuNPs conjugated with peptides against both the epidermal growth factor and transferrin receptors and loaded with the photosensitizer phthalocyanine 4 (Pc 4) have been designed and compared with monotargeted AuNPs for in vitro and in vivo studies. The (EGFpep+Tfpep)-AuNPs-Pc 4 with a particle size of ∼41 nm improved both specificity and worked synergistically to decrease time of maximal accumulation in human glioma cells that overexpressed two cell surface receptors as compared to cells that overexpressed only one. Enhanced cellular association and increased cytotoxicity were achieved. In vivo studies show notable accumulation of these agents in the brain tumor regions.

Immunosuppressive nano-therapeutic micelles downregulate endothelial cell inflammation and immunogenicity

In this study, we developed a stable, nontoxic novel micelle nanoparticle to attenuate responses of endothelial cell (EC) inflammation when subjected to oxidative stress, such as observed in organ transplantation. Targeted Rapamycin Micelles (TRaM) were synthesized using PEG-PE-amine and N-palmitoyl homocysteine (PHC) with further tailoring of the micelle using targeting peptides (cRGD) and labeling with far-red fluorescent dye for tracking during cellular uptake studies. Our results revealed that the TRaM was approximately 10 nm in diameter and underwent successful internalization in Human Umbilical Vein EC (HUVEC) lines. Uptake efficiency of TRaM nanoparticles was improved with the addition of a targeting moiety. In addition, our TRaM therapy was able to downregulate both mouse cardiac endothelial cell (MCEC) and HUVEC production and release of the pro-inflammatory cytokines, IL-6 and IL-8 in normal oxygen tension and hypoxic conditions. We were also able to demonstrate a dose-dependent uptake of TRaM therapy into biologic tissues ex vivo. Taken together, these data demonstrate the feasibility of targeted drug delivery in transplantation, which has the potential for conferring local immunosuppressive effects without systemic consequences while also dampening endothelial cell injury responses.

Delivery of a drug cache to glioma cells overexpressing platelet-derived growth factor receptor using lipid nanocarriers

AIM Glioblastoma multiforme is a devastating disease with no curative options due to the difficulty in achieving sufficient quantities of effective chemotherapies into the tumor past the blood-brain barrier. Micelles loaded with temozolomide (TMZ) were designed to increase the delivery of this drug into the brain. MATERIALS & METHODS pH-responsive micelles composed of distearoyl phosphoethanolamine-PEG-2000-amine and N-palmitoyl homocysteine were surface-functionalized with PDGF peptide and Dylight 680 fluorophore. RESULTS & CONCLUSION PDGF-micelles containing TMZ have specific uptake and increased killing in glial cells compared with untargeted micelles. In vivo studies demonstrated selective accumulation of PDGF-micelles containing TMZ in orthotopic gliomas implanted in mice. Targeted micelle-based drug carrier systems hold potential for delivery of a wide variety of hydrophobic drugs thereby reducing its systemic toxicity.

Nanotechnology Applications for Diffuse Intrinsic Pontine Glioma

Diffuse intrinsic pontine gliomas (DIPGs) are invariably fatal tumors found in the pons of elementary school aged children. These tumors are grade II-IV gliomas, with a median survival of less than 1 year from diagnosis when treated with standard of care (SOC) therapy. Nanotechnology may offer therapeutic options for the treatment of DIPGs. Multiple nanoparticle formulations are currently being investigated for the treatment of DIPGs. Nanoparticles based upon stable elements, polymer nanoparticles, and organic nanoparticles are under development for the treatment of brain tumors, including DIPGs. Targeting of nanoparticles is now possible as delivery techniques that address the difficulty in crossing the blood brain barrier (BBB) are developed. Theranostic nanoparticles, a combination of therapeutics and diagnostic nanoparticles, improve imaging of the cancerous tissue while delivering therapy to the local region. However, additional time and attention should be directed to developing a nanoparticle delivery system for treatment of the uniformly fatal pediatric disease of DIPG.

Fluorescence and Bioluminescence Imaging of Orthotopic Brain Tumors in Mice

Optical imaging strategies, such as fluorescence and bioluminescence imaging, are non-invasive, in vivo whole body imaging techniques utilized to study cancer. Optical imaging is widely used in preclinical work because of its ease of use and cost-friendliness. It also provides the opportunity to study animals and biological responses longitudinally over time. Important considerations include depth of tissue penetration, photon scattering, absorption and the choice of light emitting probe, all of which affect the resolution (image quality and data information) and the signal to noise ratio of the image. We describe how to use bioluminescence and fluorescence imaging to track a chemotherapeutic delivery nanocarrier conjugated with a fluorophore to determine its localization in vivo.

Abstract 5413: Double-targeted theranostic gold nanoparticles for treatment of brain tumors

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Therapeutic drug delivery across the blood-brain barrier (BBB) is not only inefficient but also nonspecific, thereby posing a major shortcoming in effective treatment of brain cancer. Photodynamic therapy (PDT) is a localized treatment modality, relying on both a photosensitizer and drug activation using a specific wavelength. The widespread use of PDT in brain tumor therapy has been partially hampered by non-targeted phototoxicity towards healthy tissue. The development of nanoparticles selectively targeted to cell surface receptors that can act as drug delivery vehicles is critical for improving the treatment and therapeutic responsiveness in inaccessible tumors, such as glioblastomas. Gold nanoparticles (Au NP) provide an excellent platform with a surface that can be tailored to attach biomolecules for targeted drug delivery and biocompatible coatings that can efficiently encapsulate the hydrophobic photosensitizer drug, Pc 4, thereby reducing off-site cytotoxicity. In this study, we demonstrate a novel double targeted, noncovalent Au NP drug delivery agent, which selectively delivers drugs to brain tumors for PDT. These double-targeted Au NPs loaded with Pc 4 have been compared with previously studied single targeted Au NPs. Hydrophobic Au NPs have been cap exchanged with mono- and bi-functional PEG linkers. Specific targeting of the PEGylated Au NPs to glioma cells is achieved by coupling receptor-binding peptides to the carboxyl moiety of the bi-functional PEG linker. Subsequently, hydrophobic Pc 4 is adsorbed in the PEG corona (mono-functional linker) to form Pc 4 loaded and targeted Au NPs. Packaging of Pc 4 within the PEG core impedes leaching of the drug into the extracellular environment and improves circulation in vivo. UV-Vis absorption measurements indicate encapsulation of Pc 4 within the PEGylated Au NPs. Hydrodynamic diameter of these agents lies well within the limits needed to cross the BBB as determined by dynamic light scattering. In vitro cell uptake studies in glioma cell lines, LN229 and U87, which express differential patterns of the epidermal growth factor (EGF) and transferrin (Tf) receptor targets, show a significant increase in cellular uptake and intracellular localization for double targeted conjugates as compared to either single targeted Au NPs. Titration studies have been carried out in cells to optimize delivery; the optimal concentration for double targeted Au NPs is 500 nM, half of the current clinical standard observed for single targeted Au NPs over the same period of incubation. In vivo imaging utilizing real time, longitudinal fluorescence in mice shows notable accumulation of these agents in the tumor. Co-localization of the targeted Au NPs in regions overexpressing EGF and Tf receptors has been validated by immunohistochemistry. Future experiments involve activation of Pc 4 by PDT after delivery by the double-targeted Au NPs and monitoring tumor cell death. Note: This abstract was not presented at the meeting. Citation Format: Suraj K. Dixit, Yun Zhu, Alfred Moore, Malcolm Kenney, Ann-Marie Broome. Double-targeted theranostic gold nanoparticles for treatment of brain tumors. [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 5413. doi:10.1158/1538-7445.AM2014-5413

Application of Deacetylated Poly-N-Acetyl Glucosamine Nanoparticles for the Delivery of miR-126 for the Treatment of Cecal Ligation and Puncture-Induced Sepsis

Sepsis is an acute inflammatory syndrome in response to infection. In some cases, excessive inflammation from sepsis results in endothelial dysfunction and subsequent increased vascular permeability leading to organ failure. We previously showed that treatment with endothelial progenitor cells, which highly express microRNA-126 (miR-126), improved survival in mice subjected to cecal ligation and puncture (CLP) sepsis. miRNAs are important regulators of gene expression and cell function, play a major role in endothelial homeostasis, and may represent an emerging therapeutic modality. However, delivery of miRNAs to cells in vitro and in vivo is challenging due to rapid degradation by ubiquitous RNases. Herein, we developed a nanoparticle delivery system separately combining deacetylated poly-N-acetyl glucosamine (DEAC-pGlcNAc) polymers with miRNA-126-3p and miRNA-126-5p and testing these combinations in vitro and in vivo. Our results demonstrate that DEAC-pGlcNAc polymers have an appropriate size and zeta potential for cellular uptake and when complexed, DEAC-pGlcNAc protects miRNA from RNase A degradation. Further, DEAC-pGlcNAc efficiently encapsulates miRNAs as evidenced by preventing their migration in an agarose gel. The DEAC-pGlcNAc-miRNA complexes were taken up by multiple cell types and the delivered miRNAs had biological effects on their targets in vitro including pERK and DLK-1. In addition, we found that delivery of DEAC-pGlcNAc alone or DEAC-pGlcNAc:miRNA-126-5p nanoparticles to septic animals significantly improved survival, preserved vascular integrity, and modulated cytokine production. These composite studies support the concept that DEAC-pGlcNAc nanoparticles are an effective platform for delivering miRNAs and that they may provide therapeutic benefit in sepsis.

Abstract 4467: Treating brain tumors with targeted-micelles containing rapamycin

The development of selectively targeted nanoparticles that can act as drug delivery vehicles is critical for improving the treatment and monitoring of glioblastoma, a life threatening disease. Rapamycin (Sirolimus, rapa), a large, lipophilic carboxylic lactone-lactam macrolide antibiotic, is recognized for its potent anti-proliferative and immunosuppressive effects in vitro and in vivo. These properties make rapa a potential chemotherapeutic agent against several tumors. Despite its promising properties, clinical applications of rapa have been limited due to its hydrophobicity, limiting its utility as an intravenously administered drug. Presently, the commercially available formulations of rapa include tablet or oral forms. Nevertheless, the low oral bioavailability of rapa limits the effectiveness of both of these forms. In addition, the lipophilicity makes the drug susceptible to attachment to the lipid membranes of normal as well as cancer cells. A selectively targeted carrier for rapa will enhance its delivery to malignant cells, avoid non-specific interactions, and reduce non-tumor toxicity. In order to design an efficient and effective drug carrier, we created a multifunctional nanocarrier that contains a tailored surface on the carrier to attach biomolecules for targeted drug delivery; a biocompatible coating which can efficiently encapsulate the hydrophobic drug thereby reducing cytotoxicity; and the capability for stimuli-induced (pH) disruption of the carrier agent for slow and controlled drug release to the desired environment, Micelles are the preferred choice of carrier as they fulfill these requirements based on their composition. Micelles containing rapamycin drug are synthesized using PEG-PE-Amine and N-palmitoyl homocysteine (PHC, pH sensitive lipid breaks in endosome pH 5.5). Specific targeting of the micelles to glioblastoma cells is achieved by PDGF (platelet derived growth factor) or EGF (epidermal growth factor) coupled to the amine moeity of the DSPE-PEG. In addition these micelles have been labeled with a NIR fluorphore to track them for cellular uptake. These micelles have an advantage of small size ( Note: This abstract was not presented at the meeting. Citation Format: Ann-Marie Broome, Suraj K. Dixit, Kayla Miller, Alfred Moore, Amy-Lee Bredlau. Treating brain tumors with targeted-micelles containing rapamycin. [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 4467. doi:10.1158/1538-7445.AM2014-4467

Abstract 3981: Targeted delivery of temozolomide to pediatric brain tumors using micelle-based theranostic nanocarriers

Diffuse intrinsic pontine glioma (DIPG) is the single most devastating pediatric brain tumor. Surgical excision of these tumors, due to the exquisite location of the pons, is dangerous and is not curative. No chemotherapy has been identified that can control these tumors. One theory for this failure is that the pons is a unique location into which delivery of medications at therapeutic concentrations is singularly challenging. Current standard of care utilizes temozolomide (TMZ), a pro-drug that releases a DNA alkylating agent that is used to kill glial cells. TMZ is very toxic when delivered systemically and therapeutic dosages are limited by severe side effects. These factors necessitate a selectively targeted carrier for TMZ to deliver the drug efficiently to malignant cells avoiding nonspecific interaction and reducing offsite toxicity. In order to design an efficient and effective drug carrier, we addressed several issues: a tailored surface on the carrier to attach biomolecules for targeted drug delivery; a biocompatible coating which can efficiently encapsulate the hydrophobic drug thereby reducing cytotoxicity; and stimuli-induced (pH) disruption of the carrier agent for slow and controlled drug release to the desired environment. Micelles are the preferred choice of carrier as they fulfill these requirements based on their composition. Micelles containing drug are synthesized using PEG-PE-Amine and N-palmitoyl homocysteine (pH sensitive lipid breaks in endosome pH 5.5). Specific targeting of the micelles to glioblastoma cells is achieved by coupling a short 12 a.a. PDGF (platelet derived growth factor) peptide to the amine moeity of the DSPE-PEG. In addition, these micelles have been labeled with a NIR fluorophore to track them for cellular uptake and can be used to image tumor internalization in vivo. These micelles have an advantage of small size ( Citation Format: Kayla Miller, Suraj K. Dixit, Amy-Lee Bredlau, Ann-Marie Broome. Targeted delivery of temozolomide to pediatric brain tumors using micelle-based theranostic nanocarriers. [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 3981. doi:10.1158/1538-7445.AM2014-3981