Synthetic High-density Lipoprotein Nanoparticles Research Articles

Synthetic high-density lipoprotein nanoparticles delivering rapamycin for the treatment of age-related macular degeneration

Synthetic high-density lipoprotein (sHDL) and rapamycin (Rap) have both been shown to be potential treatments for age-related macular degeneration (AMD). The low aqueous solubility of Rap, however, limits its therapeutic utility. Here we used an Apolipoprotein A-I mimetic peptide and phospholipid-based sHDL for the intravitreal delivery of Rap. By incorporation of Rap in sHDL nanoparticles (sHDL-Rap), we achieve 125-fold increase in drug aqueous concentration. When applied in vitro to retinal pigment epithelium cells, sHDL-Rap exhibited the abilities to efflux cholesterol, neutralize endotoxin, and suppress NF-κB activation. As an mTOR inhibitor, Rap induced autophagy and inhibited NF-κB-mediated pro-inflammatory signaling. Additionally, a greater reduction in lipofuscin accumulation and increased anti-inflammatory effects were achieved by sHDL-Rap relative to free drug or sHDL alone. In vivo studies demonstrated that sHDL reached the target retina pigment epithelium (RPE) layer following intravitreal administration in rats. These results suggest that sHDL-Rap holds potential as a treatment for AMD.

HDL nanoparticles have wound healing and anti-inflammatory properties and can topically deliver miRNAs.

microRNAs regulate numerous biological processes, making them potential therapeutic agents. Problems with delivery and stability of these molecules have limited their usefulness as treatments. We demonstrate that synthetic high-density lipoprotein nanoparticles (HDL NPs) topically applied to the intact ocular surface are taken up by epithelial and stromal cells. microRNAs complexed to HDL NPs (miR-HDL NPs) are similarly taken up by cells and tissues and retain biological activity. Topical treatment of diabetic mice with either HDL NPs or miR-HDL NPs significantly improved corneal re-epithelialization following wounding compared with controls. Mouse corneas with alkali burn-induced inflammation, topically treated with HDL NPs, displayed clinical, morphological and immunological improvement. These results should yield a novel HDL NP-based eye drop for patients with compromised wound healing ability (diabetics) and/or corneal inflammatory diseases (e.g. dry eye).

Synthetic high-density lipoprotein nanoparticles for the treatment of Niemann\u2013Pick diseases

BackgroundNiemann–Pick disease type C is a fatal and progressive neurodegenerative disorder characterized by the accumulation of unesterified cholesterol in late endosomes and lysosomes. We sought to develop new therapeutics for this disorder by harnessing the body’s endogenous cholesterol scavenging particle, high-density lipoprotein (HDL).MethodsHere we design, optimize, and define the mechanism of action of synthetic HDL (sHDL) nanoparticles.ResultsWe demonstrate a dose-dependent rescue of cholesterol storage that is sensitive to sHDL lipid and peptide composition, enabling the identification of compounds with a range of therapeutic potency. Peripheral administration of sHDL to Npc1 I1061T homozygous mice mobilizes cholesterol, reduces serum bilirubin, reduces liver macrophage size, and corrects body weight deficits. Additionally, a single intraventricular injection into adult Npc1 I1061T brains significantly reduces cholesterol storage in Purkinje neurons. Since endogenous HDL is also a carrier of sphingomyelin, we tested the same sHDL formulation in the sphingomyelin storage disease Niemann–Pick type A. Utilizing stimulated Raman scattering microscopy to detect endogenous unlabeled lipids, we show significant rescue of Niemann–Pick type A lipid storage.ConclusionsTogether, our data establish that sHDL nanoparticles are a potential new therapeutic avenue for Niemann–Pick diseases.

Mimetic sHDL nanoparticles: A novel drug-delivery strategy to target triple-negative breast cancer

BackgroundWithanolides are naturally derived heat shock protein 90 inhibitors that are potent in preclinical models of triple negative breast cancers. Conjugation to synthetic high-density lipoprotein nanoparticles improves solubility and targets delivery to the scavenger receptor B1. Triple negative breast cancers highly overexpress the scavenger receptor B1, and we hypothesize that encapsulation of the novel withalongolide A 4,19,27-triacetate by synthetic high-density lipoprotein will have enhanced efficacy against triple negative breast cancers in vivo. MethodsValidated human triple negative breast cancer cell lines were evaluated for the scavenger receptor B1 expression by quantitative polymerase chain reaction and Western blot. Withalongolide A 4,19,27-triacetate inhibitory concentration50 values were obtained using CellTiter-Glo assays (Promega, Madison, WI, USA). The scavenger receptor B1–mediated drug uptake was evaluated in vitro under fluorescence microscopy and in vivo with IVIS imaging of mouse xenografts (MD-MBA-468LN). To evaluate drug efficacy, mice were treated with synthetic high-density lipoprotein alone, withalongolide A 4,19,27-triacetate alone, withalongolide A 4,19,27-triacetate synthetic high-density lipoprotein, and chemotherapy or Prussian blue stain (control). ResultsTriple negative breast cancer cell lines had greater scavenger receptor B1 expression by quantitative polymerase chain reaction and Western blot versus controls. Fluorescent-labeled synthetic high-density lipoprotein uptake was scavenger receptor B1–mediated in vitro, and in vivo tumor uptake using IVIS imaging demonstrated significantly increased tumor radiant efficiency versus control. Inhibitory concentration50 for withalongolide A 4,19,27-triacetate–treated cells with or without synthetic high-density lipoprotein encapsulation were 70-fold to 200-fold more potent than synthetic high-density lipoprotein alone. In triple negative breast cancer mouse xenografts, treatment with synthetic high-density lipoprotein withalongolide A 4,19,27-triacetate resulted in a 54% decrease in tumor volume compared with the control or with synthetic high-density lipoprotein alone. ConclusionThe synthetic high-density lipoprotein withalongolide A 4,19,27-triacetate nanoconjugates are potent against triple negative breast cancers and show improved scavenger receptor B1–mediated targeting. Treatment with synthetic high-density lipoprotein–encapsulated withalongolide A 4,19,27-triacetate is able to significantly decrease the growth of tumor in mice compared with the control and has better efficacy than the current standard of care, warranting further evaluation as a novel therapeutic agent.

Synthetic high-density lipoprotein nanoconjugate targets neuroblastoma stem cells, blocking migration and self-renewal

Pathways critical for neuroblastoma cancer stem cell function are targeted by 4,19,27-triacetyl withalongolide A (WGA-TA). Because neuroblastoma cells and their cancer stem cells highly overexpress the scavenger receptor class B type 1 receptor that binds to synthetic high-density lipoprotein, we hypothesized that a novel mimetic synthetic high-density lipoprotein nanoparticle would be an ideal carrier for the delivery of 4,19,27-triacetyl withalongolide to neuroblastoma and neuroblastoma cancer stem cells. Expression of scavenger receptor class B type 1 in validated human neuroblastoma cells was evaluated by quantitative polymerase chain reaction (qPCR) and Western blot. In vitro cellular uptake of synthetic high-density lipoprotein nanoparticles was observed with a fluorescence microscope. In vivo biodistribution of synthetic high-density lipoprotein nanoparticles was investigated with IVIS imaging. Self-renewal and migration/invasion were assessed by sphere formation and Boyden chamber assays, respectively. Viability was analyzed by CellTiter-Glo assay. Cancer stem cell markers were evaluated by flow cytometry. qPCR and Western blot analysis revealed a higher level of scavenger receptor class B type 1 expression and drug uptake in N-myc amplified neuroblastoma cells. In vitro uptake of synthetic high-density lipoprotein was almost completely blocked by excess synthetic high-density lipoprotein. The synthetic high-density lipoprotein nanoparticles mainly accumulated in the tumor and liver, but not in other organs. Synthetic HDL-4,19,27-triacetyl withalongolide showed a 1,000-fold higher potency than the carrier (synthetic high-density lipoprotein) alone (P < .01) to kill neuroblastoma cells. Additionally, a dose-dependent decrease in sphere formation, invasion, migration, and cancer stem cell markers was observed after treatment of neuroblastoma cells with synthetic high-density lipoprotein-4,19,27-triacetyl withalongolide A. Synthetic high-density lipoprotein is a promising platform to improve the delivery of anticancer drug 4,19,27-triacetyl withalongolide A to neuroblastomas and neuroblastoma cancer stem cells through SR-B1 targeting in vitro and in vivo.

Effect of Synthetic High Density Lipoproteins Modification with Polyethylene Glycol on Pharmacokinetics and Pharmacodynamics.

Synthetic high density lipoprotein nanoparticles (sHDLs) capable of mobilizing excess cholesterol from atherosclerotic arteries and delivering it to the liver for elimination have been shown to reduce plaque burden in patients. Unfortunately, sHDLs have a narrow therapeutic index and relative to the endogenous HDL shorter circulation half-life. Surface modification with polyethylene glycol (PEG) was investigated for its potential to extend sHDL circulation in vivo. Various amounts (2.5, 5, and 10%) and different chain lengths (2 and 5 kDa) of PEG-modified lipids were incorporated in sHDL's lipid membrane. Incorporating PEG did not reduce the ability of sHDL to facilitate cholesterol efflux, nor did it inhibit cholesterol uptake by the liver cells. By either adding more PEG or using PEG of longer chain lengths, the circulation half-life was extended. Addition of PEG also increased the area under the curve for the phospholipid component of sHDL (p < 0.05), but not for the apolipoprotein A-I peptide component of sHDL, suggesting sHDL is remodeled by endogenous lipoproteins in vivo. The extended phospholipid circulation led to a higher mobilization of plasma free cholesterol, a biomarker for facilitation of reverse cholesterol transport. The area under the cholesterol mobilization increased about 2-4-fold (p < 0.05), with greater increases observed for longer PEG chains and higher molar percentages of incorporated PEGylated lipids. Mobilized cholesterol was associated primarily with the HDL fraction, led to a transient increase in VLDL cholesterol, and returned to baseline 24 h postdose. Overall, PEGylation of sHDL led to beneficial changes in sHDL particle pharmacokinetic and pharmacodynamic behaviors.

Effect of size and pegylation of liposomes and peptide-based synthetic lipoproteins on tumor targeting

Synthetic high-density lipoprotein nanoparticles (sHDL) are a valuable class of nanomedicines with established animal safety profile, clinical tolerability and therapeutic efficacy for cardiovascular applications. In this study we examined how the scavenger receptor B-I-mediated (SR-BI) tumor-targeting ability of sHDL, long plasma circulation half-life, and small particle size (9.6±0.2nm) impacted sHDL accumulation in SR-BI positive colorectal carcinoma cells, 3D tumor spheroids, and in vivo xenografts. We compared tumor accumulation of sHDL with that of liposomes (LIP, 130.7±0.8nm), pegylated liposomes (PEG-LIP, 101±2nm), and pegylated sHDL (12.1±0.1nm), all prepared with the same lipid components. sHDL penetrated deep (210μm) into tumor spheroids and exhibited 12- and 3-fold higher in vivo solid tumor accumulation, compared with LIP (p<0.01) and PEG-LIP (p<0.05), respectively. These results suggest that sHDL with established human safety possess promising intrinsic tumor-targeted properties.

Scavenger Receptor Type B1 Is Essential for High Density Lipoprotein Nanoparticle Induced B-Cell Lymphoma Cell Death

Introduction:We recently reported that biomimetic, synthetic high-density lipoprotein nanoparticles (HDL NP), similar to natural HDL in size, shape, charge, and composition potently induce apoptosis in human B cell lymphoma cell lines in vitro, and in vivo, without adversely affecting primary hepatocytes or macrophages (Yang et al. 2013. PNAS 110(7): 2511-6). We showed that HDL NPs bind to the high-affinity HDL receptor, scavenger receptor type B-1 (SR-B1), expressed by lymphoma cells, and may play a functional role in apoptosis of B cell lymphomas.Methods:We analyzed tissue microarrays (TMAs) from patients with non-Hodgkin's lymphoma (NHL) for SR-B1 expression. In silico analyses of gene microarray datasets from the publicly available database Oncomine were conducted to investigate SR-B1 expression in lymphoma at the mRNA level. The B cell lymphoma cell lines Ramos (Burkitt's lymphoma), SUDHL4 (diffuse large B cell lymphoma) and HF-1 (transformed follicular lymphoma) were used to investigate the requirement for SR-B1 in HDL NP induced cell death. A blocking antibody, and stably expressed shRNA targeting SR-B1 expression were used to inhibit HDL NP interactions with SR-B1.Results:SR-B1 was overexpressed in a subpopulation of NHL represented in available TMAs. At the mRNA level, SR-B1 was up-regulated in 20% of lymphoma data sets (6 of 30). These data further confirm SR-B1 as a potential target of therapeutic intervention in B cell lymphomas.Antibody blockade of SR-B1 in the SR-B1+ cell lines Ramos, SUDHL4, and HF-1 prevented HDL NP induced cell death in a dose dependent manner (Figure 1A). Stable knockdown of SR-B1 by shRNA in Ramos cells also protected against HDL NP induced cell death compared with wild type and scrambled shRNA controls (Figure 1B). HDL NP induced cholesterol efflux in Ramos, SUDHL4, and HF-1 cells was reduced by the SR-B1 blocking antibody, or SR-B1 knockdown, further supporting that the HDL NP binds SR-B1.Conclusion:SR-B1 is expressed in primary B cell lymphomas, and interference of HDL NP interaction with SR-B1, through antibody blockade or knockdown of SR-B1, abrogated HDL NP induced cell death in multiple B cell lymphoma cell lines. Taken together, our data demonstrate the requirement of SR-B1 in HDL NP induced lymphoma cell death, and provide a rationale to pursue HDL NPs as potent therapy for B cell lymphomas in cases that express SR-B1. [Display omitted] DisclosuresGordon:Dr Leo I. Gordon: Patents & Royalties: Patent for gold nanoparticles pending; Northwestern University: Employment. Thaxton:Aurasense: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: High Density Lipoprotein Nanoparticles for Lymphoma.

Synthetic high-density lipoprotein nanoparticles: A novel therapeutic strategy for adrenocortical carcinomas

Chemotherapeutic strategies for adrenocortical carcinoma (ACC) carry substantial toxicities. Cholesterol is critical for ACC cell growth and steroidogenesis, and ACC cells overexpress scavenger receptor BI, which uptakes cholesterol from circulating high-density lipoprotein (HDL) cholesterol. We hypothesize that cholesterol-free synthetic-HDL nanoparticles (sHDL) will deplete cholesterol and synergize with chemotherapeutics to achieve enhanced anticancer effects at lesser (less toxic) drug levels. The antiproliferative efficacy of ACC cells for the combinations of sHDL with chemotherapeutics was tested by Cell-Titer Glo. Cortisol levels were measured from the culture media. Effects on steroidogenesis was measured by real-time polymerase chain reaction (RT-PCR). Induction of apoptosis was evaluated by flow cytometry. Combination Index (CI) for sHDL and either etoposide (E), cisplatin (P), or mitotane (M) demonstrated synergy (CI < 1) for antiproliferation. Alone or in combination with the chemotherapy drugs, sHDL was able to decrease cortisol production by 70-90% compared with P alone or controls (P < .01). RT-PCR indicated inhibition of steroidogenic enzymes for sHDL (P < .01 vs no sHDL). Combination therapy with sHDL increased apoptosis by 30-50% compared with drug or sHDL alone (P < .03), confirmed by a decrease in the mitochondrial potential. sHDL can act synergistically and lessen the amount of M/E/P needed for anticancer efficacy in ACC in part owing to cholesterol starvation. This novel treatment strategy warrants further investigation translationally.

Abstract 3669: Engineering the biointerface of synthetic high density lipoprotein nanoparticles enables efficient nucleic acid loading, delivery, and target gene regulation in cancer cells

Abstract Local treatment of cancer confined to the primary organ can be curative. However, other than a few success stories, locally advanced and metastatic cancer remains incurable. Ideally, new therapies are needed that specifically target cancer cells and derive efficacy by targeting molecular pathways that are unique to distinct tumor profiles and individual patients. Toward this end, we focus on the use of biomimicry to exploit mechanisms by which cancer cells uptake molecular cargo. Naturally, some cancer cells are dependent upon exogenous cholesterol for cellular signaling, growth, and in some cancers, such as prostate, steroidogenesis. However, cholesterol is a highly hydrophobic molecule that requires a delivery vehicle for systemic circulation and cellular delivery. High-density lipoproteins (HDLs) are natural nanostructures that engage cancer cells to deliver exogenous cholesterol. In addition to cholesterol, HDLs bind and deliver nucleic acids, such as microRNAs. Importantly, HDLs are targeted to a surface receptor, scavenger receptor type B-1 (SR-B1), whereupon binding allows for particle uptake and cargo delivery. SR-B1 has been shown to be overexpressed in human cancers, thus making HDLs a highly attractive delivery vehicle for therapeutic nucleic acid cargo. Because of the propensity of HDL to bind, stabilize, and deliver RNA our group has developed ways to synthesize lipoproteins nanoparticles and use them as therapeutic RNA delivery vehicles. Importantly, natural HDLs are highly dynamic species, which frequently undergo exchange of molecular surface components other lipoprotein species, as well as, HDLs. Recent work is focused on chemically tailoring the surface of synthetic HDL nanoparticles (HDL NPs) to understand RNA binding, stabilization, and cellular delivery. In addition, manipulating the surface chemical features of HDL also provides a way to better understand intermolecular exchange with native serum lipoproteins. Data demonstrate that by tailoring the surface chemistry of the HDL NP one can modulate the efficiency by which siRNAs can be incorporated onto the particle surface and delivered to target cells to reduce target gene expression in cancer cells. Further, data show the tendency for nanoparticle lipids to transfer to natural HDLs; however, siRNA formulated on the surface of the HDL NP remains bound, even when exposed to serum for prolonged periods. In short, exploration of HDL NPs for the systemic delivery of siRNAs may be a promising approach for targeted patient-specific cancer therapies. Citation Format: Kaylin M. McMahon, Don Vander Griend, Olga Volpert, C. Shad Thaxton. Engineering the biointerface of synthetic high density lipoprotein nanoparticles enables efficient nucleic acid loading, delivery, and target gene regulation in cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3669. doi:10.1158/1538-7445.AM2015-3669

Abstract 3671: Loading and molecular labeling of cell-specific exosomes by HDL-like AuNPs

Abstract Exosomes are 30-100 nm diameter lipid vesicles produced by most cells. Exosomes carry diverse cargo, such as RNA and proteins. Cancer cells produce an abundant amount of exosomes, which are released from cells of the primary tumor into the tumor microenvironment and the circulation. Data show that exosomes can promote cancer progression and metastasis by delivering, for instance, pro-metastatic factors to pre-metastatic sites. It is not currently possible to specifically separate tumor-derived exosomes from complex matrices. This ability would provide a better understanding of their contribution to tumorigenesis and cancer progression. Furthermore, the targeting properties of exosomes make them appealing vehicles for encapsulating and delivering therapeutic cargo. However, it is challenging to isolate and then load exosomes with specific drugs. Uniquely, our data reveal that synthetic high-density lipoprotein nanoparticles (HDL NPs) can be tailored to achieve cell-specific exosome labeling and loading of therapeutic cargo. HDL NPs are synthesized using a 5 nm diameter spherical gold nanoparticle. The gold NP serves as a template to control size and shape. Apolipoprotein A-I and phospholipids are conjugated to the gold surface, yielding a synthetic HDL NP similar in size, shape, surface chemistry, and function to some natural HDLs. Our data demonstrate that, upon addition of HDL NPs to cultured CWR 22Rv1 prostate cancer cells, they are packaged into exosomes that are secreted from these cells. Furthermore, HDL NPs made using phospholipids functionalized with either biotin or fluorophore molecules leads to the incorporation of the phospholipids into the exosomes. Thus, the specific cell-derived exosomes are labeled with gold, a molecular fluorophore label, and/or biotin. In each case, the specific exosomes are easy to isolate using methods such as fluorescent isolation or by the robust streptavidin-biotin interaction. Isolated exosomes retain the same size and molecular markers as exosomes from untreated cells. Overall, this unique method may provide control over exosomes for a myriad of diagnostic and therapeutic uses. Citation Format: Nicholas L. Angeloni, Kaylin M. McMahon, C. Shad Thaxton. Loading and molecular labeling of cell-specific exosomes by HDL-like AuNPs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3671. doi:10.1158/1538-7445.AM2015-3671

Biomimetic, synthetic HDL nanostructures for lymphoma

New therapies that challenge existing paradigms are needed for the treatment of cancer. We report a nanoparticle-enabled therapeutic approach to B-cell lymphoma using synthetic high density lipoprotein nanoparticles (HDL-NPs). HDL-NPs are synthesized using a gold nanoparticle template to control conjugate size and ensure a spherical shape. Like natural HDLs, biomimetic HDL-NPs target scavenger receptor type B-1, a high-affinity HDL receptor expressed by lymphoma cells. Functionally, compared with natural HDL, the gold NP template enables differential manipulation of cellular cholesterol flux in lymphoma cells, promoting cellular cholesterol efflux and limiting cholesterol delivery. This combination of scavenger receptor type B-1 binding and relative cholesterol starvation selectively induces apoptosis. HDL-NP treatment of mice bearing B-cell lymphoma xenografts selectively inhibits B-cell lymphoma growth. As such, HDL-NPs are biofunctional therapeutic agents, whose mechanism of action is enabled by the presence of a synthetic nanotemplate. HDL-NPs are active in B-cell lymphomas and potentially, other malignancies or diseases of pathologic cholesterol accumulation.