Aditi Jhaveri

The effect of dual ligand-targeted micelles on the delivery and efficacy of poorly soluble drug for cancer therapy

Abstract We prepared and evaluated transferrin (Tf) and monoclonal antibody (mAb) 2C5-modified dual ligand-targeted poly(ethylene glycol)–phosphatidylethanolamine micelles loaded with a poorly soluble drug, R547 (a selective adenosine triphosphate-competitive cyclin-dependent kinase inhibitor) for enhancement of targeting efficiency and cytotoxicity in vitro and in vivo to A2780 ovarian carcinoma compared to single ligand-targeted micelles. Micellar solubilization significantly improved the solubility of R547 from 1 to 800 μg/mL. The size of modified and non-modified micelles was 13–16 nm. Flow cytometry indicated significantly enhanced cellular association of dual ligand-targeted micelles compared to single ligand-targeted micelles. Confocal microscopy confirmed the Tf receptor-mediated endocytosis of rhodamine-labeled Tf-modified micelles after staining the micelle-treated cells with the endosomal marker Tf–Alexa488. The optimized dual-targeted micelles enhanced cytotoxicity in vitro against A2780 ovarian cancer cells compared to plain and single ligand-targeted micelles. Interestingly, in vivo anti-tumor efficacy was more pronounced for the preparation with a single-targeting ligand (Tf). The specific combination Tf and mAb 2C5 did not yield the expected increase in efficacy as was observed in vitro. This observation suggests that the relationships between targeting ligands in vivo could be more complex than in simplified in vitro systems, and the results of the optimization process should always be verified in vivo.

Multifunctional polymeric micelles for delivery of drugs and siRNA

Polymeric micelles, self-assembling nano-constructs of amphiphilic copolymers with a core-shell structure have been used as versatile carriers for delivery of drugs as well as nucleic acids. They have gained immense popularity owing to a host of favorable properties including their capacity to effectively solubilize a variety of poorly soluble pharmaceutical agents, biocompatibility, longevity, high stability in vitro and in vivo and the ability to accumulate in pathological areas with compromised vasculature. Moreover, additional functions can be imparted to these micelles by engineering their surface with various ligands and cell-penetrating moieties to allow for specific targeting and intracellular accumulation, respectively, to load them with contrast agents to confer imaging capabilities, and incorporating stimuli-sensitive groups that allow drug release in response to small changes in the environment. Recently, there has been an increasing trend toward designing polymeric micelles which integrate a number of the above functions into a single carrier to give rise to “smart,” multifunctional polymeric micelles. Such multifunctional micelles can be envisaged as key to improving the efficacy of current treatments which have seen a steady increase not only in hydrophobic small molecules, but also in biologics including therapeutic genes, antibodies and small interfering RNA (siRNA). The purpose of this review is to highlight recent advances in the development of multifunctional polymeric micelles specifically for delivery of drugs and siRNA. In spite of the tremendous potential of siRNA, its translation into clinics has been a significant challenge because of physiological barriers to its effective delivery and the lack of safe, effective and clinically suitable vehicles. To that end, we also discuss the potential and suitability of multifunctional polymeric micelles, including lipid-based micelles, as promising vehicles for both siRNA and drugs.

Intracellular delivery of nanocarriers and targeting to subcellular organelles

Introduction: Recent trends in drug delivery indicate a steady increase in the use of targeted therapeutics to enhance the specific delivery of biologically active payloads to diseased tissues while avoiding their off-target effects. However, in most cases, the distribution of therapeutics inside cells and their targeting to intracellular targets still presents a formidable challenge. The main barrier to intracellular delivery is the translocation of therapeutic molecules across the cell membrane, and ultimately through the membrane of their intracellular target organelles. Another prerequisite for an efficient intracellular localization of active molecules is their escape from the endocytic pathway. Areas covered: Pharmaceutical nanocarriers have demonstrated substantial advantages for the delivery of therapeutics and offer elegant platforms for intracellular delivery. They can be engineered with both intracellular and organelle-specific targeting moieties to deliver encapsulated or conjugated cargoes to specific sub-cellular targets. In this review, we discuss important aspects of intracellular drug targeting and delivery with a focus on nanocarriers modified with various ligands to specifically target intracellular organelles. Expert opinion: Intracellular delivery affords selective localization of molecules to their target site, thus maximizing their efficacy and safety. The advent of novel nanocarriers and targeting ligands as well as exploration of alternate routes for the intracellular delivery and targeting has prompted extensive research, and promises an exciting future for this field.

Targeted Transferrin-Modified Polymeric Micelles: Enhanced Efficacy in Vitro and in Vivo in Ovarian Carcinoma

In this study, transferrin (Tf)-modified poly(ethylene glycol)-phosphatidylethanolamine (mPEG-PE) micelles loaded with the poorly water-soluble drug, R547 (a potent and selective ATP-competitive cyclin-dependent kinase (CDK) inhibitor), were prepared and evaluated for their targeting efficiency and cytotoxicity in vitro and in vivo to A2780 ovarian carcinoma cells, which overexpress transferrin receptors (TfR). At 10 mM lipid concentration, both Tf-modified and plain micelles solubilized 800 μg of R547. Tf-modified micelles showed enhanced interaction with A2780 ovarian carcinoma cells in vitro. The involvement of TfR in endocytosis of Tf-modified micelles was confirmed by colocalization studies of micelle-treated cells with the endosomal marker Tf-Alexa488. We confirmed endocytosis of micelles in an intact form with micelles loaded with a fluorescent dye and additionally labeled with fluorescent lipid. The in vitro cytotoxicity and in vivo tumor growth inhibition studies in A2780-tumor bearing mice confirmed the enhanced efficacy of Tf-modified R547-loaded micelles compared to free drug solution and to nonmodified micelles. The results of this study demonstrate the potential application of Tf-conjugated polymeric micelles in the treatment of tumors overexpressing TfR.

Immunomicelles for advancing personalized therapy.

Personalized medicine, which ultimately seeks to afford tailored therapeutic regimens for individual patients, is quickly emerging as a new paradigm in the diagnosis and treatment of diseases. The idea of casting aside generic treatments in favor of patient-centric therapies has become feasible owing to advances in nanotechnology and drug delivery coupled with an enhanced knowledge of genomics and an understanding of disease at the molecular level. This review highlights polymeric immunomicelles as a class of nanocarriers that have the potential to combine diagnosis, targeted drug therapy, as well as imaging and monitoring of therapeutic response, to render a personalized approach to the management of disease. Smart multi-functional immunomicelles, as the next generation of nanocarriers, are poised for facilitating personalized cancer treatment. This review provides an assessment of immunomicelles as tools for advancing personalized therapy of diseases, with cancer being the major focus.

Combination Nanopreparations of a Novel Proapoptotic Drug - NCL-240, TRAIL and siRNA.

PurposeTo develop a multifunctional nanoparticle system carrying a combination of pro-apoptotic drug, NCL-240, TRAIL [tumor necrosis factor-α (TNF-α)-related apoptosis-inducing ligand] and anti-survivin siRNA and to test the combination preparation for anti-cancer effects in different cancer cells.MethodsPolyethylene glycol-phosphoethanolamine (PEG-PE) – based polymeric micelles were prepared carrying NCL-240. These micelles were used in combination with TRAIL-conjugated micelles and anti-survivin siRNA-S-S-PE containing micelles. All the micelles were characterized for size, zeta potential, and drug encapsulation efficiency. Different cancer cells were used to study the cytotoxicity potential of the individual as well as the combination formulations. Other cell based assays included cellular association studies of transferrin-targeted NCL-240 micelles and study of cellular survivin protein downregulation by anti-survivin siRNA-S-S-PE containing micelles.ResultsNCL-240 micelles and the combination NCL-240/TRAIL micelles significantly increased cytotoxicity in the resistant strains of SKOV-3, MCF-7 and A549 as compared to free drugs or single drug formulations. The NCL-240/TRAIL micelles were also more effective in NCI/ADR-RES cancer cell spheroids. Anti-survivin siRNA micelles alone displayed a dose-dependent reduction in survivin protein levels in A2780 cells. Treatment with NCL-240/TRAIL after pre-incubation with anti-survivin siRNA inhibited cancer cell proliferation. Additionally, a single multifunctional system composed of NCL-240/TRAIL/siRNA PM also had significant cytotoxic effects in vitro in multiple cell lines.ConclusionThese results demonstrate the efficacy of a combination of small-molecule PI3K inhibitors, TRAIL, and siRNA delivered by micellar preparations in multiple cancer cell lines.

Transferrin and octaarginine modified dual-functional liposomes with improved cancer cell targeting and enhanced intracellular delivery for the treatment of ovarian cancer

Abstract Off-target effects of drugs severely limit cancer therapy. Targeted nanocarriers are promising to enhance the delivery of therapeutics to tumors. Among many approaches for active tumor-targeting, arginine-rich cell penetrating peptides (AR-CPP) and ligands specific to target over-expressed receptors on cancer-cell surfaces, are popular. Earlier, we showed that the attachment of an AR-CPP octaarginine (R8) to the surface of DOXIL® (Doxorubicin encapsulated PEGylated liposomes) improved cytoplasmic and nuclear DOX delivery that enhanced the cytotoxic effect in vitro and improved therapeutic efficacy in vivo. Here, we report on DOX-loaded liposomes, surface-modified with, R8 and transferrin (Tf) (Dual DOX-L), to improve targeting of A2780 ovarian carcinoma cells via the over-expressed transferrin receptors (TfRs) with R8-mediated intracellular DOX delivery. Flow cytometry analysis with fluorescently labeled DualL (without DOX) showed two-fold higher cancer-cell association than other treatments after 4 h treatment. Blocking entry pathways of R8 (macropinocytosis) and Tf (receptor-mediated endocytosis, RME) resulted in a decreased cancer-cell association of DualL. Confocal microscopy confirmed involvement of both entry pathways and cytoplasmic liposome accumulation with nuclear DOX delivery for Dual DOX-L. Dual DOX-L exhibited enhanced cytotoxicity in vitro and was most effective in controlling tumor growth in vivo in an A2780 ovarian xenograft model compared to other treatments. A pilot biodistribution study showed improved DOX accumulation in tumors after Dual DOX-L treatment. All results collectively presented a clear advantage of the R8 and Tf combination to elevate the therapeutic potential of DOX-L by exploiting TfR over-expression imparting specificity followed by endosomal escape and intracellular delivery via R8.

Transferrin-targeted, resveratrol-loaded liposomes for the treatment of glioblastoma

&NA; Glioblastomas (GBMs) are highly aggressive brain tumors with a very grim prognosis even after multi‐modal therapeutic regimens. Conventional chemotherapeutic agents frequently lead to drug resistance and result in severe toxicities to non‐cancerous tissues. Resveratrol (RES), a natural polyphenol with pleiotropic health benefits, has proven chemopreventive effects in all the stages of cancer including initiation, promotion and progression. However, the poor physico‐chemical properties of RES severely limit its use as a free drug. In this study, RES was loaded into PEGylated liposomes (RES‐L) to counter its drawbacks as a free drug. Since transferrin receptors (TfRs) are up‐regulated in GBM, the liposome surface was modified with transferrin moieties (Tf‐RES‐L) to make them cancer cell‐specific. The liposomal nanomedicines developed in this project were aimed at enhancing the physico‐chemical properties of RES and exploiting the passive and active targeting capabilities of liposomes to effectively treat GBM. The RES‐L were stable, had a good drug‐loading capacity, prolonged drug‐release in vitro and were easily scalable. Flow cytometry and confocal microscopy were used to study the association with, and internalization of, Tf‐L into U‐87 MG cells. The Tf‐RES‐Ls were significantly more cytotoxic and induced higher levels of apoptosis accompanied by activation of caspases 3/7 in GBM cells when compared to free RES or RES‐L. The ability of RES to arrest cells in the S‐phase of the cell cycle, and selectively induce production of reactive oxygen species in cancer cells were probably responsible for its cytotoxic effects. The therapeutic efficacy of RES formulations was evaluated in a subcutaneous xenograft mouse model of GBM. A tumor growth inhibition study and a modified survival study showed that Tf‐RES‐Ls were more effective than other treatments in their ability to inhibit tumor growth and improve survival in mice. Overall, the liposomal nanomedicines of RES developed in this project exhibited favorable in vitro and in vivo efficacies, which warrant their further investigation for the treatment of GBMs.

Targeting energy metabolism of cancer cells: Combined administration of NCL-240 and 2-DG

Cancer cells increase their metabolism to produce the energy and biomolecules necessary for growth and proliferation. Thus, energy metabolism pathways may serve as targets for anti-cancer therapy. NCL-240 is a second generation anti-cancer drug belonging to the PITenins class of PI3K-Akt inhibitors. Our analysis suggested that NCL-240 caused disruptions in mitochondrial oxidative phosphorylation and up-regulated glycolysis, as evidenced by the loss of NMR peaks for the amino acid products derived from the TCA cycle along with presence of only lactate peaks and the loss of glucose peaks. NCL-240 was combined with 2-deoxy-d-glucose (2-DG) in early proof-of-concept studies on multiple cell lines. 2-DG enhanced cell death response to NCL-240 administration, with cytotoxicity results similar to those under hypoglycemic conditions. In further studies, NCL-240 encapsulated in phosphatidylcholine/cholesterol liposomes was combined with freely dissolved 2-DG. Cell cycle analysis of sensitive and resistant strains of A2780 cells treated with combinations of NCL-240/2-DG pointed to a G0/G1 phase arrest for 80-90% of the total, indicating an inability to grow and divide. Cytotoxicity studies with in vitro cancer cell monolayer models confirmed the results of cell cycle analysis. Significant improvements in cytotoxicity with combination treatments over control and individual treatments were seen in multiple cell lines. NCI/ADR-RES cancer cell spheroids further demonstrated the effectiveness of a NCL-240/2-DG combination.

Abstract 3251: Evaluating the efficacy of transferrin-targeted, resveratrol-loaded liposomes in treating glioblastoma

Purpose: This study evaluated the efficacy of a natural polyphenol resveratrol (RES) loaded into liposomes (RES-L) which are modified with transferrin (Tf) (Tf-RES-L), to specifically direct it to glioblastoma (GBM) cells. RES is known to act on both the bulk tumor cells (BTCs) as well as the highly resistant tumor-initiating cell (TIC) population within GBMs. However, low aqueous solubility, chemical instability, poor pharmacokinetics and low bioavailability severely limit its use as a free drug. We developed RES-L with an aim to counter these drawbacks and to eradicate the BTC and TIC populations in GBMs. Since both these sub-populations also over-express Tf receptors, we exploited this feature for the target-specific delivery of Tf-RES-L in GBM. We hypothesize that Tf-RES-L will show an improved efficacy versus the free drug or non-targeted liposomes and will act as an efficacious platform for delivery of RES. Methods: The neurosphere (NS) assay was used to develop TIC models using GBM cell lines. The NS were characterized using in vitro limiting dilution assays (LDA) and expression of the surface marker CD133. RES-Ls were prepared by thin-film rehydration method and Tf was attached to the liposomes using an in-house conjugation protocol. Liposomes were characterized for their size, charge, morphology and drug-loading efficacy. Rhodamine labeled, Tf-targeted formulations were tested for their association with and internalization into cells using flow cytometry and confocal microscopy, respectively. Cytotoxicity assays, oxidative stress measurements, apoptosis assays and cell-cycle analyses were carried out for the GBM monolayers and NS cultures. In vivo tumor-inhibition studies were carried out using a mouse model of GBM. Results: NS cultures showed the presence of TICs as determined from LDAs and CD-133 expression. RES inhibited the anchorage-independent growth of GBM NS. All RES formulations induced a time and dose-dependent cytotoxicity in cells. At low concentrations, Tf-RES-Ls were significantly more cytotoxic compared to free RES or RES-L. RES formulations arrested GBM cells in the S-phase of the cell-cycle at low concentrations and exhibited a pro-oxidant effect at higher concentrations inducing significant oxidative stress only in GBM cells but not in primary human astrocytes. Tf-RES-L induced significantly higher levels of apoptosis accompanied by activation of caspases 3/7 in GBM cells compared to the free drug and non-targeted RES-L. The Tf- targeted formulations also associated with and internalized into GBM cells significantly better than the non-targeted counterparts. Conclusions: RES effectively eliminates both, the BTCs and TICs in glioblastoma, and its encapsulation in Tf-modified liposomes still further improves its efficacy compared to the free drug or drug in non-targeted liposomes. Tf-RES-Ls thus seem like a very promising nanomedicine candidate for further development to treat GBM. Citation Format: Aditi Jhaveri, Vladimir Torchilin. Evaluating the efficacy of transferrin-targeted, resveratrol-loaded liposomes in treating glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3251. doi:10.1158/1538-7445.AM2017-3251