A Spontaneous Assembling Lipopeptide Nanoconjugate Transporting the Anthracycline Drug N-Benzyladriamycin-14-valerate for Personalized Therapy of Ewing Sarcoma.

To meet the current need for a tumor-selective, targeted therapy regimen associated with reduced toxicity, our laboratory has developed a spontaneously assembled nanostructure that resembles high-density lipoproteins (HDLs). These myristoyl-5A (MYR-5A) nanotransporters are designed to safely transport lipophilic pharmaceuticals, including a novel anthracycline drug (N-benzyladriamycin-14-valerate (AD198)). This formulation has been found to enhance the therapeutic efficacy and reduced toxicity of drugs in preclinical studies of 2D and 3D models of Ewing sarcoma (EWS) and cardiomyocytes. Our findings indicate that the MYR-5A/AD198 nanocomplex delivers its payload selectively to cancer cells via the scavenger receptor type B1 (SR-B1), thus providing a solid proof of concept for the development of an improved and highly effective, potentially personalized therapy for EWS while protecting against treatment-associated cardiotoxicity.

Mannose-Coated Reconstituted Lipoprotein Nanoparticles for the Targeting of Tumor-Associated Macrophages: Optimization, Characterization, and In Vitro Evaluation of Effectiveness.

Reconstituted high-density lipoprotein nanoparticles (rHDL NPs) have been utilized as delivery vehicles to a variety of targets, including cancer cells. However, the modification of rHDL NPs for the targeting of the pro-tumoral tumor-associated macrophages (TAMs) remains largely unexplored. The presence of mannose on nanoparticles can facilitate the targeting of TAMs which highly express the mannose receptor at their surface. Here, we optimized and characterized mannose-coated rHDL NPs loaded with 5,6-dimethylxanthenone-4-acetic acid (DMXAA), an immunomodulatory drug. Lipids, recombinant apolipoprotein A-I, DMXAA, and different amounts of DSPE-PEG-mannose (DPM) were combined to assemble rHDL-DPM-DMXAA NPs. The introduction of DPM in the nanoparticle assembly altered the particle size, zeta potential, elution pattern, and DMXAA entrapment efficiency of the rHDL NPs. Collectively, the changes in physicochemical characteristics of rHDL NPs upon the addition of the mannose moiety DPM indicated that the rHDL-DPM-DMXAA NPs were successfully assembled. The rHDL-DPM-DMXAA NPs induced an immunostimulatory phenotype in macrophages pre-exposed to cancer cell-conditioned media. Furthermore, rHDL-DPM NPs delivered their payload more readily to macrophages than cancer cells. Considering the effects of the rHDL-DPM-DMXAA NPs on macrophages, the rHDL-DPM NPs have the potential to serve as a drug delivery platform for the selective targeting of TAMs.

Lipoproteins and the Tumor Microenvironment.

The tumor microenvironment (TME) plays a key role in enhancing the growth of malignant tumors and thus contributing to "aggressive phenotypes," supporting sustained tumor growth and metastasis. The precise interplay between the numerous components of the TME that contribute to the emergence of these aggressive phenotypes is yet to be elucidated and currently under intense investigation. The purpose of this article is to identify specific role(s) for lipoproteins as part of these processes that facilitate (or oppose) malignant growth as they interact with specific components of the TME during tumor development and treatment. Because of the scarcity of literature reports regarding the interaction of lipoproteins with the components of the tumor microenvironment, we were compelled to explore topics that were only tangentially related to this topic, to ensure that we have not missed any important concepts.

Probing the Assembly of HDL Mimetic, Drug Carrying Nanoparticles Using Intrinsic Fluorescence.

Reconstituted high-density lipoprotein (HDL) containing apolipoprotein A-I (Apo A-I) mimics the structure and function of endogenous (human plasma) HDL due to its function and potential therapeutic utility in atherosclerosis, cancer, neurodegenerative diseases, and inflammatory diseases. Recently, a new class of HDL mimetics has emerged, involving peptides with amino acid sequences that simulate the the primary structure of the amphipathic alpha helices within the Apo A-I protein. The findings reported in this communication were obtained using a similar amphiphilic peptide (modified via conjugation of a myristic acid residue at the amino terminal aspartic acid) that self-assembles (by itself) into nanoparticles while retaining the key features of endogenous HDL. The studies presented here involve the macromolecular assembly of the myristic acid conjugated peptide (MYR-5A) into nanomicellar structures and its characterization via steady-state and time-resolved fluorescence spectroscopy. The structural differences between the free peptide (5A) and MYR-5A conjugate were also probed, using tryptophan fluorescence, FÓ§rster resonance energy transfer (FRET), dynamic light scattering, and gel exclusion chromatography. To our knowledge, this is the first report of a lipoprotein assembly generated from a single ingredient and without a separate lipid component. The therapeutic utility of these nanoparticles (due to their capablity to incorporate a wide range of drugs into their core region for targeted delivery) was also investigated by probing the role of the scavenger receptor type B1 in this process. SIGNIFICANCE STATEMENT: Although lipoproteins have been considered as effective drug delivery agents, none of these nanoformulations has entered clinical trials to date. A major challenge to advancing lipoprotein-based formulations to the clinic has been the availability of a cost-effective protein or peptide constituent, needed for the assembly of the drug/lipoprotein nanocomplexes. This report of a robust, spontaneously assembling drug transport system from a single component could provide the template for a superior, targeted drug delivery strategy for therapeutics of cancer and other diseases (Counsell and Pohland, 1982).

The SR-B1 Receptor as a Potential Target for Treating Glioblastoma.

PURPOSE: The goal of these studies was to provide proof of concept for a novel targeted therapy for Glioblastoma Multiforme (GBM). Methods. These studies involve the evaluation of reconstituted high density lipoprotein (rHDL) nanoparticles (NPs) as delivery agents for the drug, mammalian Target of Rapamycin (mTOR) inhibitor Everolimus (EVR) to GBM cells. Cytotoxicity studies and assessment of downstream effects, including apoptosis, migration, and cell cycle events, were probed, in relation to the expression of scavenger receptor B type 1 (SR-B1) by GBM cells. RESULTS: Findings from cytotoxicity studies indicate that the rHDL/EVR formulation was 185 times more potent than free EVR against high SR-B1 expressing cell line (LN 229). Cell cycle analysis revealed that rHDL/EVR treated LN229 cells had a 5.8 times higher apoptotic cell population than those treated with EVR. The sensitivity of GBM cells to EVR treatment was strongly correlated with SR-B1 expression. CONCLUSIONS: These studies present strong proof of concept regarding the efficacy of delivering EVR and likely other agents, via a biocompatible transport system, targeted to the SR-B1 receptor that is upregulated in most cancers, including GBM. Targeting the SR-B1 receptor could thus lead to effective personalized therapy of GBM.

Identifying and targeting angiogenesis-related microRNAs in ovarian cancer.

Current anti-angiogenic therapy for cancer is based mainly on inhibition of the vascular endothelial growth factor pathway. However, due to the transient and only modest benefit from such therapy, additional approaches are needed. Deregulation of microRNAs (miRNAs) has been demonstrated to be involved in tumor angiogenesis and offers opportunities for a new therapeutic approach. However, effective miRNA-delivery systems are needed for such approaches to be successful. In this study, miRNA profiling of patient data sets, along with in vitro and in vivo experiments, revealed that miR-204-5p could promote angiogenesis in ovarian tumors through THBS1. By binding with scavenger receptor class B type 1 (SCARB1), reconstituted high-density lipoprotein-nanoparticles (rHDL-NPs) were effective in delivering miR-204-5p inhibitor (miR-204-5p-inh) to tumor sites to suppress tumor growth. These results offer a new understanding of miR-204-5p in regulating tumor angiogenesis.

[99mTc-HYNIC-N-dodecylamide]: a new hydrophobic tracer for labelling reconstituted high-density lipoproteins (rHDL) for radioimaging.

Despite the widespread use of nanotechnology in radio-imaging applications, lipoprotein based delivery systems have received only limited attention so far. These studies involve the synthesis of a novel hydrophobic radio-imaging tracer consisting of a hydrazinonicotinic acid (HYNIC)-N-dodecylamide and 99mTc conjugate that can be encapsulated into rHDL nanoparticles (NPs). These rHDL NPs can selectively target the Scavenger Receptor type B1 (SR-B1) that is overexpressed on most cancer cells due to excess demand for cholesterol for membrane biogenesis and thus can target tumors in vivo. We provide details of the tracer synthesis, characterization of the rHDL/tracer complex, in vitro uptake, stability studies and in vivo application of this new radio-imaging approach.

Reconstituted HDL: Drug Delivery Platform for Overcoming Biological Barriers to Cancer Therapy.

Drug delivery to malignant tumors is limited by several factors, including off-target toxicities and suboptimal benefits to cancer patient. Major research efforts have been directed toward developing novel technologies involving nanoparticles (NPs) to overcome these challenges. Major obstacles, however, including, opsonization, transport across cancer cell membranes, multidrug-resistant proteins, and endosomal sequestration of the therapeutic agent continue to limit the efficiency of cancer chemotherapy. Lipoprotein-based drug delivery technology, "nature's drug delivery system," while exhibits highly desirable characteristics, it still needs substantial investment from private/government stakeholders to promote its eventual advance to the bedside. Consequently, this review focuses specifically on the synthetic (reconstituted) high-density lipoprotein rHDL NPs, evaluating their potential to overcome specific biological barriers and the challenges of translation toward clinical utilization and commercialization. This highly robust drug transport system provides site-specific, tumor-selective delivery of anti-cancer agents while reducing harmful off-target effects. Utilizing rHDL NPs for anti-cancer therapeutics and tumor imaging revolutionizes the future strategy for the management of a broad range of cancers and other diseases.

Lipoproteins for therapeutic delivery: recent advances and future opportunities.

The physiological role(s) of mammalian plasma lipoproteins is to transport hydrophobic molecules (primarily cholesterol and triacylglycerols) to their respective destinations. Lipoproteins have also been studied as drug-delivery agents due to their advantageous payload capacity, long residence time in the circulation and biocompatibility. The purpose of this review is to briefly discuss current findings with the focus on each type of formulation's potential for clinical applications. Regarding utilizing lipoprotein type formulation for cancer therapeutics, their potential for tumor-selective delivery is also discussed.

Scavenger receptor class B type 1 regulates neuroblastoma cell proliferation, migration and invasion.

Neuroblastoma (NB) is an extra cranial pediatric embryonal tumor most prevalent in children less than 1 year of age. NB accounts for 7% of all pediatric cancers but accounts for 15% of all childhood cancer deaths. Scavenger receptor class B type 1 (SR-B1), a mediator of cellular cholesterol uptake, is overexpressed in and have been linked to the aggressiveness of many cancers. Nevertheless, no studies have so far investigated the relationship between SR-B1 and NB. Elucidation of receptors that promote NB may pave the way for discovery of new therapeutic targets. Here we show that inhibition of SR-B1 reduced cell survival, migration and invasion, and cholesterol content in NB cell lines. Additionally analysis of SR-B1 levels in NB patient biopsies using the R2: Genomics Analysis and Visualization Platform showed that high SR-B1 expression correlated with decreased overall and event-free survival.

Ultrasound-Stimulated Drug Delivery Using Therapeutic Reconstituted High-Density Lipoprotein Nanoparticles.

The abnormal tumor vasculature and the resulting abnormal microenvironment are major barriers to optimal chemotherapeutic drug delivery. It is well known that ultrasound (US) can increase the permeability of the tumor vessel walls and enhance the accumulation of anticancer agents. Reconstituted high-density lipoproteins (rHDL) nanoparticles (NPs) allow selective delivery of anticancer agents to tumor cells via their overexpressed scavenger receptor type B1 (SR-B1) receptor. The goal of this study is to investigate the potential of noninvasive US therapy to further improve delivery and tumor uptake of the payload from rHDL NPs, preloaded with an infrared dye (IR-780), aimed to establish a surrogate chemotherapeutic model with optical localization. Athymic nude mice were implanted orthotopically with one million breast cancer cells (MDA-MB-231/Luc). Three weeks later, animals were divided into seven groups with comparable mean tumor size: control, low, moderate, and high concentration of rHDL NPs alone groups, as well as these three levels of rHDL NPs plus US therapy groups (N = 7 to 12 animals per group), where low, moderate and high denote 5, 10, and 50 µg of the IR-780 dye payload per rHDL NP injection, respectively. The US therapy system included a single element focused transducer connected in series with a function generator and power amplifier. A custom 3D printed cone with an acoustically transparent aperture and filled with degassed water allowed delivery of focused US energy to the tumor tissue. US exposure involved a pulsed sequence applied for a duration of 5 min. Each animal in the US therapy groups received a slow bolus co-injection of MB contrast agent and rHDL NPs. Animals were imaged using a whole-body optical system to quantify intratumoral rHDL NP accumulation at baseline and again at 1 min, 30 min, 24 h, and 48 h. At 48 h, all animals were euthanized and tumors were excised for ex vivo analysis. We investigated a noninvasive optical imaging method for monitoring the effects of US-stimulated drug delivery of IR-780 dye-loaded rHDL NPs in living animals. No change in optical imaging data was found in the control animals. However, there was considerable dye accumulation (surrogate drug) within 48 h in the low (5 µg), moderate (10 µg), and high (50 µg) rHDL NP concentration-dosed group animals (p < 0.09). With US therapy added to the experimental protocol, there was an additional and significant increase in local tumor drug uptake at 48 h (p < 0.02). Optical image data collected from ex vivo tumor samples confirmed tumor retention of the IR-780 dye-loaded rHDL NPs and correlated positively with in vivo optical imaging results (R2 > 0.69, p < 0.003). IR-780 dye extraction from the tumor tissue samples confirmed the in vivo and ex vivo US therapy findings. Overall, the addition of US therapy considerably improved local rHDL NP accumulation in tumor tissue. This study concludes that US-mediated drug delivery can facilitate tumor uptake of rHDL NPs and more research is warranted to optimize the drug dosing schedule and the respective therapeutic protocols.

SR-B1-targeted nanodelivery of anti-cancer agents: a promising new approach to treat triple-negative breast cancer.

Patients with triple-negative breast cancer (TNBC) have a considerably less favorable prognosis than those with hormone-positive breast cancers. TNBC patients do not respond to current endocrine treatment and have a 5-year survival prognosis of <30%. The research presented here is intended to fill a void toward the much needed development of improved treatment strategies for metastatic TNBC. The overall goal of this research was to evaluate the effectiveness of reconstituted high-density lipoprotein (rHDL) nanoparticles (NPs) as delivery agents for anti-TNBC drugs. Using lapatinib and valrubicin as components of the rHDL/drug complexes resulted in a significantly better performance of the NP-transported drugs compared with their free (unencapsulated) counterparts. The enhancement of the therapeutic effect and the protection of normal cells (cardiomyocytes) achieved via the rHDL NPs were likely due to the overexpression of the high-density lipoprotein (HDL) (scavenger receptor class B type 1 [SR-B1]) receptor by the TNBC cells.

Fluorescence properties of doxorubicin in PBS buffer and PVA films.

We studied steady-state and time-resolved fluorescence properties of an anticancer drug Doxorubicin in a saline buffer and poly-vinyl alcohol (PVA) film. Absorption of Doxorubicin, located at blue-green spectral region, allows a convenient excitation with visible light emitting diodes or laser diodes. Emission of Doxorubicin with maximum near 600nm can be easily detected with photomultipliers and CCD cameras. Both, absorption and fluorescence spectra in polymeric matrix show more pronounced vibronic structures than in solution. Also, the steady-state anisotropy in the polymer film is significantly higher than in the saline solution. In PVA film the fluorescence anisotropy is about 0.30 whereas in the saline buffer only 0.07. Quantum efficiencies of Doxorubicin were compared to a known standard Rhodamine 101 which has fluorescence emission in a similar spectral region. The quantum yield of Doxorubicin in PVA film is more than 10% and about twice higher than in the saline solution. Similarly, the lifetime of doxorubicin in PVA film is about 2ns whereas in the saline solution only about 1ns. The fluorescence anisotropy decays show that Doxorubicin molecules are freely rotating in the saline buffer with a correlation time of about 290ps, and are almost completely immobilized in the PVA film. The spectroscopic investigations presented in this manuscript are important, as they provide answers to changes in molecular properties of Doxorubicin depending changes in the local environment, which is useful when synthesizing nanoparticles for Doxorubicin entrapment.

Superparamagnetic reconstituted high-density lipoprotein nanocarriers for magnetically guided drug delivery.

Current cancer chemotherapy is frequently associated with short- and long-term side effects, affecting the quality of life of cancer survivors. Because malignant cells are known to overexpress specific surface antigens, including receptors, targeted drug delivery is often utilized to reduce or overcome side effects. The current study involves a novel targeting approach using specifically designed nanoparticles, including encapsulation of the anti-cancer drug valrubicin into superparamagnetic iron oxide nanoparticle (SPION) containing reconstituted high-density lipoprotein (rHDL) nanoparticles. Specifically, rHDL-SPION-valrubicin hybrid nanoparticles were assembled and characterized with respect to their physical and chemical properties, drug entrapment efficiency and receptor-mediated release of the drug valrubicin from the nanoparticles to prostate cancer (PC-3) cells. Prussian blue staining was used to assess nanoparticle movement in a magnetic field. Measurements of cytotoxicity toward PC-3 cells showed that rHDL-SPION-valrubicin nanoparticles were up to 4.6 and 31 times more effective at the respective valrubicin concentrations of 42.4 µg/mL and 85 µg/mL than the drug valrubicin alone. These studies showed, for the first time, that lipoprotein drug delivery enhanced via magnetic targeting could be an effective chemotherapeutic strategy for prostate cancer.

Targeting the SR-B1 Receptor as a Gateway for Cancer Therapy and Imaging.

Malignant tumors display remarkable heterogeneity to the extent that even at the same tissue site different types of cells with varying genetic background may be found. In contrast, a relatively consistent marker the scavenger receptor type B1 (SR-B1) has been found to be consistently overexpressed by most tumor cells. Scavenger Receptor Class B Type I (SR-BI) is a high density lipoprotein (HDL) receptor that facilitates the uptake of cholesterol esters from circulating lipoproteins. Additional findings suggest a critical role for SR-BI in cholesterol metabolism, signaling, motility, and proliferation of cancer cells and thus a potential major impact in carcinogenesis and metastasis. Recent findings indicate that the level of SR-BI expression correlate with aggressiveness and poor survival in breast and prostate cancer. Moreover, genomic data show that depending on the type of cancer, high or low SR-BI expression may promote poor survival. This review discusses the importance of SR-BI as a diagnostic as well as prognostic indicator of cancer to help elucidate the contributions of this protein to cancer development, progression, and survival. In addition, the SR-B1 receptor has been shown to serve as a potential gateway for the delivery of therapeutic agents when reconstituted high density lipoprotein nanoparticles are used for their transport to cancer cells and tumors. Opportunities for the development of new technologies, particularly in the areas of cancer therapy and tumor imaging are discussed.

Photophysical characterization of anticancer drug valrubicin in rHDL nanoparticles and its use as an imaging agent.

Nanoparticles are target-specific drug delivery agents that are increasingly used in cancer therapy to enhance bioavailability and to reduce off target toxicity of anti-cancer agents. Valrubicin is an anti-cancer drug, currently approved only for vesicular bladder cancer treatment because of its poor water solubility. On the other hand, valrubicin carrying reconstituted high density lipoprotein (rHDL) nanoparticles appear ideally suited for extended applications, including systemic cancer chemotherapy. We determined selected fluorescence properties of the free (unencapsulated) drug vs. valrubicin incorporated into rHDL nanoparticles. We have found that upon encapsulation into rHDL nanoparticles the quantum yield of valrubicin fluorescence increased six fold while its fluorescence lifetime increased about 2 fold. Accordingly, these and potassium iodide (KI) quenching data suggest that upon incorporation, valrubicin is localized deep in the interior of the nanoparticle, inside the lipid matrix. Fluorescence anisotropy of the rHDL valrubicin nanoparticles was also found to be high along with extended rotational correlation time. The fluorescence of valrubicin could also be utilized to assess its distribution upon delivery to prostate cancer (PC3) cells. Overall the fluorescence properties of the rHDL: valrubicin complex reveal valuable novel characteristics of this drug delivery vehicle that may be particularly applicable when used in systemic (intravenous) therapy.

HDL as a drug and nucleic acid delivery vehicle.

This review is intended to evaluate the research findings and potential clinical applications of drug transport systems, developed based on the concepts of the structure/function and physiological role(s) of high density lipoprotein type nanoparticles. These macromolecules provide targeted transport of cholesteryl esters (a highly lipophilic payload) in their natural/physiological environment. The ability to accommodate highly water insoluble constituents in their core regions enables High density lipoproteins (HDL) type nanoparticles to effectively transport hydrophobic drugs subsequent to systemic administration. Even though the application of reconstituted HDL in the treatment of a number of diseases is reviewed, the primary focus is on the application of HDL type drug delivery agents in cancer chemotherapy. The use of both native and synthetic HDL as drug delivery agents is compared to evaluate their respective potentials for commercial and clinical development. The current status and future perspectives for HDL type nanoparticles are discussed, including current obstacles and future applications in therapeutics.

A homodimeric BODIPY rotor as a fluorescent viscosity sensor for membrane-mimicking and cellular environments.

Fluorescence properties of a novel homodimeric BODIPY dye rotor for Fluorescence Lifetime Imaging Microscopy (FLIM) are reported. Steady state and time resolved fluorescence measurements established the viscosity dependent behaviour in vitro. Homodimeric BODIPY embedded in different membrane mimicking lipid vesicles (DPPC, POPC and POPC plus cholesterol) is demonstrated to be a viable sensor for fluorescence lifetime based viscosity measurements. Moreover, SKOV3 cells readily endocytosed the dye, which accumulated in membranous structures inside the cytoplasm thereby allowing viscosity mapping of internal cell components.

Targeted nanoparticles for pediatric leukemia therapy.

The two major forms of leukemia, acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), account for about one-third of the malignancies diagnosed in children. Despite the marked successes in ALL and AML treatment, concerns remain regarding the occurrence of resistant disease in subsets of patients, the residual effects of therapy that often persist for decades beyond the cessation of treatment. Therefore, new approaches are needed to reduce or to avoid off target toxicities, associated with chemotherapy and their long-term residual effects. Recently, nanotechnology has been employed to enhance cancer therapy, via improving the bioavailability and therapeutic efficacy of anti-cancer agents. While in the last several years, numerous review articles appeared detailing the size, composition, assembly, and performance evaluation of different types of drug carrying nanoparticles, the description and evaluation of lipoprotein-based drug carriers have been conspicuously absent from most of these major reviews. The current review focuses on such information regarding nanoparticles with an emphasis on high density lipoprotein-based drug delivery systems to examine their potential role(s) in the enhanced treatment of children with leukemia.

The potential role of nanotechnology in therapeutic approaches for triple negative breast cancer.

Triple Negative Breast Cancer, TNBC, a highly aggressive and metastatic type of breast cancer, is characterized by loss of expression of the estrogen receptor (ER), progesterone receptor (PR), and a lack of overexpression of the human epidermal growth factor receptor 2 (HER2). It is a heterogeneous group of tumors with diverse histology, molecular uniqueness and response to treatment. Unfortunately, TNBC patients do not benefit from current anti-HER2 or hormone positive targeted breast cancer treatments; consequently, these patients rely primarily on chemotherapy. However, the 5-year survival rate for woman with metastatic TNBC is less than 30%. As a result of ineffective treatments, TNBC tumors often progress to metastatic lesions in the brain and lung. Brain metastases of invasive breast cancer are associated with 1 and 2 year survival rate of 20% and <2% respectively. Because the only current systemic treatment for TNBC is chemotherapy, alternative targeted therapies are urgently needed to improve the prognosis for TNBC patients. This review is focused on opportunities for developing new approaches for filling the current void in an effective treatment for TNBC patients.