High-density Lipoprotein Nanoparticles Research Articles

Photophysical Characterization and In Vitro Evaluation of α-Mangostin-Loaded HDL Mimetic Nano-Complex in LN-229 Glioblastoma Spheroid Model.

Cytotoxic activity has been reported for the xanthone α-mangostin (AMN) against Glioblastoma multiforme (GBM), an aggressive malignant brain cancer with a poor prognosis. Recognizing that AMN's high degree of hydrophobicity is likely to limit its systemic administration, we formulated AMN using reconstituted high-density lipoprotein (rHDL) nanoparticles. The photophysical characteristics of the formulation, including fluorescence lifetime and steady-state anisotropy, indicated that AMN was successfully incorporated into the rHDL nanoparticles. To our knowledge, this is the first report on the fluorescent characteristics of AMN with an HDL-based drug carrier. Cytotoxicity studies in a 2D culture and 3D spheroid model of LN-229 GBM cells and normal human astrocytes showed an enhanced therapeutic index with the rHDL-AMN formulation compared to the unincorporated AMN and Temozolomide, a standard GBM chemotherapy agent. Furthermore, treatment with the rHDL-AMN facilitated a dose-dependent upregulation of autophagy and reactive oxygen species generation to a greater extent in LN-229 cells compared to astrocytes, indicating the reduced off-target toxicity of this novel formulation. These studies indicate the potential therapeutic benefits to GBM patients via selective targeting using the rHDL-AMN formulation.

Designer high-density lipoprotein particles enhance endothelial barrier function and suppress inflammation.

High-density lipoprotein (HDL) nanoparticles promote endothelial cell (EC) function and suppress inflammation, but their utility in treating EC dysfunction has not been fully explored. Here, we describe a fusion protein named ApoA1-ApoM (A1M) consisting of apolipoprotein A1 (ApoA1), the principal structural protein of HDL that forms lipid nanoparticles, and ApoM, a chaperone for the bioactive lipid sphingosine 1-phosphate (S1P). A1M forms HDL-like particles, binds to S1P, and is signaling competent. Molecular dynamics simulations showed that the S1P-bound ApoM moiety in A1M efficiently activated EC surface receptors. Treatment of human umbilical vein ECs with A1M-S1P stimulated barrier function either alone or cooperatively with other barrier-enhancing molecules, including the stable prostacyclin analog iloprost, and suppressed cytokine-induced inflammation. A1M-S1P injection into mice during sterile inflammation suppressed neutrophil influx and inflammatory mediator secretion. Moreover, systemic A1M administration led to a sustained increase in circulating HDL-bound S1P and suppressed inflammation in a murine model of LPS-induced endotoxemia. We propose that A1M administration may enhance vascular endothelial barrier function, suppress cytokine storm, and promote resilience of the vascular endothelium.

Mannose-functionalization of reconstituted high-density lipoprotein nanoparticles improves payload delivery and enhances M2-to-M1 phenotype reprogramming of RAW 264.7 macrophages polarized by B16-F10 melanoma cells

The targeting and conversion of the immunosuppressive (M2) tumor-associated macrophages (TAMs) to an immunostimulatory (M1) phenotype can induce tumor regression in advanced melanoma. We have previously characterized and reported the ability of reconstituted high-density lipoprotein nanoparticles (rHDL NPs) functionalized with DSPE-PEG-mannose (DPM) to deliver payload to macrophages. Herein, we investigate the modulation of macrophage phenotype and payload delivery mechanisms of the rHDL-DPM NPs in RAW 264.7 murine macrophages exposed to the conditioned medium (CM) from murine B16-F10 melanoma cells. The rHDL-DPM NPs loaded with the Stimulator of Interferon genes agonist, DMXAA, reduced protein levels of M2 markers. Through the mannose moiety, the rHDL-DPM-DMXAA NPs enhanced the production of interferon β and CXCL10 compared to the free DMXAA in the B16-F10 CM-educated RAW 264.7 macrophages. Compared to their non-mannosylated counterpart, the rHDL-DPM NPs delivered their payload more efficiently to the B16-F10 CM-educated RAW 264.7 macrophages. Mechanistically, both the scavenger receptor type B class 1 (SR-B1) and the mannose receptor (CD206) facilitated payload delivery to the macrophages via endocytic and non-endocytic mechanisms. Finally, the CM from rHDL-DPM-DMXAA NPs -treated macrophages enhanced paclitaxel (paclitaxel)-mediated cytotoxicity in B16-F10 cells. Together, these in vitro findings demonstrate the potential of the mannose-functionalized rHDL NPs in improving the targeting of M2-like TAMs and treatment outcomes when combined with immunotherapy or PTX in B16-F10 melanoma in vivo models.

Effects of lipoprotein nanoparticles' composition and size on their internalization in plant and mammalian cells.

The internalization of engineered high-density lipoprotein nanoparticles (engineered lipoproteins [eLPs]) with different lipid and protein compositions, zeta potentials, and/or sizes were analyzed in representative plant and mammalian cells. The impact of the addition of a cell-penetrating peptide to eLPs on the internalization was very small in Bright Yellow (BY)-2 protoplasts compared with HeLa cells. When eLPs were prepared with one of the abundant lipids in BY-2 cells, digalactosyldiacylglycerol (DGDG) (eLP4), its internalization was dramatically increased only in HeLa cells. Such an increase in HeLa cells was also obtained for liposomes containing DGDG in a DGDG content-dependent manner. Increasing the size and zeta potential of eLPs improved their internalization in both HeLa cells and in BY-2 protoplasts but to quite varying degrees. Although eLPs tended to stay at the plasma membrane (PM) in BY-2 protoplasts with much less internalization, the PM-bound eLPs somehow promoted the internalization of coexisting nanobeads in cell culture media. These results provide fundamental insight into the future design of lipid nanoparticles for drug delivery in mammalian and plant cells.

Biokinetics, radiopharmacokinetics and estimation of the absorbed dose in healthy organs due to Technetium-99m transported in the core and on the surface of reconstituted high-density lipoprotein nanoparticles

The development of rHDL-radionuclide theragnostic systems requires evaluation of the absorbed doses that would be produced in healthy tissues and organs at risk. Technetium-99m is the most widely used radionuclide for diagnostic imaging, therefore, the design of theragnostic reconstituted high density-lipoprotein (rHDL) nanosystems labeled with Technetium-99m offers multiple possibilities. Objective: To determine the biokinetics, radiopharmacokinetics and estimate the absorbed doses induced in healthy organs by Technetium-99m transported in the core and on the surface of rHDL. Methods: Biokinetic and radiopharmacokinetic models of rHDL/[99mTc]Tc-HYNIC-DA (Technetium-99m in the core) and [99mTc]Tc-HYNIC-rHDL (Technetium-99m on the surface) were calculated from their ex vivo biodistribution in healthy mice. Absorbed doses were estimated by the MIRD formalism using OLINDA/EXM and LMFIT softwares. Results: rHDL/[99mTc]Tc-HYNIC-DA and [99mTc]Tc-HYNIC-rHDL show instantaneous absorption in kidney, lung, heart and pancreas, with slower absorption in spleen. rHDL/[99mTc]Tc-HYNIC-DA is absorbed more slowly in the intestine, while [99mTc]Tc-HYNIC-rHDL is absorbed more slowly in the liver. The main target organ for rHDL/[99mTc]Tc-HYNIC-DA, which is hydrophobic in nature, is the liver, whereas the kidney is for the more hydrophilic [99mTc]Tc-HYNIC-rHDL. Assuming that 925 MBq (25 mCi) of Technetium-99m, carried in the core or on the surface of rHDL, are administered, the maximum tolerated doses for the organs of greatest accumulation are not exceeded. Conclusion: Theragnostic systems based on 99mTc-labeled rHDL are safe from the dosimetric point of view. The dose estimates obtained can be used to adjust the 99mTc-activity to be administered in future clinical trials.

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.

Bosutinib high density lipoprotein nanoformulation has potent tumour radiosensitisation effects

Disruption of the cell cycle is among the most effective approach to increase tumour cells’ radio-sensitivity. However, the presence of dose-limiting side effects hampers the clinical use of tyrosine kinase inhibitors targeting the cell cycle. Towards addressing this challenge, we identified a bosutinib nanoformulation within high density lipoprotein nanoparticles (HDL NPs) as a promising radiosensitiser. Bosutinib is a kinase inhibitor clinically approved for the treatment of chronic myeloid leukemia that possesses radiosensitising properties through cell cycle checkpoint inhibition. We found that a remarkably high bosutinib loading (> 10%) within HDL NPs could be reliably achieved under optimal preparation conditions. The radiosensitisation activity of the bosutinib-HDL nanoformulation was first assessed in vitro in UM-SCC-1 head and neck squamous cell carcinoma (HNSCC) cells, which confirmed efficient disruption of the radiation induced G2/M cell cycle arrest. Interestingly, the bosutinib nanoformulation out-performed free bosutinib, likely because of the specific affinity of HDL NPs with tumour cells. The combination of bosutinib-HDL NPs and radiotherapy significantly controlled tumour growth in an immunocompetent murine HNSCC model. The bosutinib-HDL nanoformulation also enhanced the radiation induced immune response through the polarisation of tumour associated macrophages towards proinflammatory phenotypes.

Salinomycin-Loaded High-Density Lipoprotein Exerts Promising Anti-Ovarian Cancer Effects by Inhibiting Epithelial-Mesenchymal Transition.

BackgroundEffective treatments for ovarian cancer remain elusive, and survival rates have long been considered grim. Ovarian cancer stem cells (OCSCs) and epithelial–mesenchymal transition (EMT) are associated with cancer progression and metastasis, as well as drug resistance and eventual treatment failure. Salinomycin (Sal) has an extensive effect on a variety of cancer stem cells (CSCs); however, its poor water solubility and toxicity to healthy tissues at high doses limit further research into its potential as an anti-cancer drug. We proposed a therapeutic strategy by constructing a tumor-targeting carrier that mimics high-density lipoprotein (HDL) to synthesize salinomycin-loaded high-density lipoprotein (S-HDL). This strategy helps reduce the side effects of salinomycin, thereby improving its clinical benefits.MethodsOCSCs were isolated from ovarian cancer cells (OCCs) and the uptake of HDL nanoparticles was observed using laser confocal microscopes. After the cell viability analysis revealed the inhibitory effect of S-HDL on OCCs and OCSCs, the main biological processes influenced by S-HDL were predicted with a transcriptome sequencing analysis and verified in vitro and in vivo.ResultsCellular uptake analysis showed that the HDL delivery system was able to significantly enhance the uptake of Sal by OCCs, tentatively validating the targeting role of recombinant HDL, so that S-HDL could reduce the toxicity of Sal and increase its anti-ovarian cancer effects. Conversely, S-HDL could exert anti-ovarian cancer effects by inhibiting the proliferation of OCCs and OCSCs, promoting apoptosis, blocking EMT, and suppressing stemness and angiogenesis-related protein expression in vitro and in vivo.ConclusionS-HDL had stronger anti-ovarian cancer effects than unencapsulated Sal. Thus, it may be a potential agent for ovarian cancer treatment in the future.

Development of activated endothelial targeted high-density lipoprotein nanoparticles.

Endothelial inflammation is an important pathophysiological driving force in various acute and chronic inflammatory diseases. High-density lipoproteins (HDLs) play critical roles in regulating endothelial functions and resolving endothelial inflammation. In the present study, we developed synthetic HDLs (sHDLs) which actively target inflamed endothelium through conjugating vascular cell adhesion protein 1 (VCAM-1) specific VHPK peptide. The active targeting of VHPK-sHDLs was confirmed in vitro on TNF-α activated endothelial cells. VHPK-sHDLs presented potent anti-inflammatory efficacies in vitro through the reduction of proinflammatory cytokine production and inhibition of leukocyte adhesion to activated endothelium. VHPK-sHDLs showed increased binding on inflamed vessels and alleviated LPS-induced lung inflammation in vivo. The activated endothelium-targeted sHDLs may be further optimized to resolve endothelial inflammation in various inflammatory diseases.

Effect of Lipid Composition on the Atheroprotective Properties of HDL-Mimicking Micelles.

Atherosclerosis progression is driven by an imbalance of cholesterol and unresolved local inflammation in the arteries. The administration of recombinant apolipoprotein A-I (ApoA-I)-based high-density lipoprotein (HDL) nanoparticles has been used to reduce the size of atheroma and rescue inflammatory response in clinical studies. Because of the difficulty in producing large quantities of recombinant ApoA-I, here, we describe the preparation of phospholipid-based, ApoA-I-free micelles that structurally and functionally resemble HDL nanoparticles. Micelles were prepared using various phosphatidylcholine (PC) lipids combined with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol)-2000] (DSPE-PEG2k) to form nanoparticles of 15–30 nm in diameter. The impacts of PC composition and PEGylation on the anti-inflammatory activity, cholesterol efflux capacity, and cholesterol crystal dissolution potential of micelles were investigated in vitro. The effects of micelle composition on pharmacokinetics and cholesterol mobilization ability were evaluated in vivo in Sprague Dawley rats. The study shows that the composition of HDL-mimicking micelles impacts their overall atheroprotective properties and supports further investigation of micelles as a therapeutic for the treatment of atherosclerosis.

89Zr-Labeled High-Density Lipoprotein Nanoparticle PET Imaging Reveals Tumor Uptake in Patients with Esophageal Cancer.

Nanomedicine holds promise for the delivery of therapeutic and imaging agents to improve cancer treatment outcomes. Preclinical studies have demonstrated that high-density lipoprotein (HDL) nanoparticles accumulate in tumor tissue on intravenous administration. Whether this HDL-based nanomedicine concept is feasible in patients is unexplored. Using a multimodal imaging approach, we aimed to assess tumor uptake of exogenously administered HDL nanoparticles in patients with esophageal cancer. Methods: The HDL mimetic CER-001 was radiolabeled using 89Zr to allow for PET/CT imaging. Patients with primary esophageal cancer staged T2 and above were recruited for serial 89Zr-HDL PET/CT imaging before starting chemoradiation therapy. In addition, patients underwent routine 18F-FDG PET/CT and 3-T MRI scanning (diffusion-weighted imaging/intravoxel incoherent motion imaging and dynamic contrast-enhanced MRI) to assess tumor glucose metabolism, tumor cellularity and microcirculation perfusion, and tumor vascular permeability. Tumor biopsies were analyzed for the expression of HDL scavenger receptor class B1 and macrophage marker CD68 using immunofluorescence staining. Results: Nine patients with adenocarcinoma or squamous cell carcinoma underwent all study procedures. After injection of 89Zr-HDL (39.2 ± 1.2 [mean ± SD] MBq), blood-pool SUVmean decreased over time (11.0 ± 1.7, 6.5 ± 0.6, and 3.3 ± 0.5 at 1, 24, and 72 h, respectively), whereas liver and spleen SUVmean remained relatively constant (4.1 ± 0.6, 4.0 ± 0.8, and 4.3 ± 0.8 at 1, 24, and 72 h, respectively, for the liver; 4.1 ± 0.3, 3.4 ± 0.3, and 3.1 ± 0.4 at 1, 24, and 72 h, respectively, for the spleen) and kidney SUVmean markedly increased over time (4.1 ± 0.9, 9.3 ± 1.4, and 9.6 ± 2.0 at 1, 24, and 72 h, respectively). Tumor uptake (SUVpeak) increased over time (3.5 ± 1.1 and 5.5 ± 2.1 at 1 and 24 h, respectively [P = 0.016]; 5.7 ± 1.4 at 72 h [P = 0.001]). The effective dose of 89Zr-HDL was 0.523 ± 0.040 mSv/MBq. No adverse events were observed after the administration of 89Zr-HDL. PET/CT and 3-T MRI measures of tumor glucose metabolism, tumor cellularity and microcirculation perfusion, and tumor vascular permeability did not correlate with tumor uptake of 89Zr-HDL, suggesting that a specific mechanism mediated the accumulation of 89Zr-HDL. Immunofluorescence staining of clinical biopsies demonstrated scavenger receptor class B1 and CD68 positivity in tumor tissue, establishing a potential cellular mechanism of action. Conclusion: To our knowledge, this was the first 89Zr-HDL study in human oncology. 89Zr-HDL PET/CT imaging demonstrated that intravenously administered HDL nanoparticles accumulated in tumors of patients with esophageal cancer. The administration of 89Zr-HDL was safe. These findings may support the development of HDL nanoparticles as a clinical delivery platform for drug agents. 89Zr-HDL imaging may guide drug development and serve as a biomarker for individualized therapy.

Formulation of simvastatin within high density lipoprotein enables potent tumour radiosensitisation.

Preclinical, clinical and epidemiologic studies have established the potent anticancer and radiosensitisation effects of HMG-CoA reductase inhibitors (statins). However, the low bioavailability of oral statin formulations is a key barrier to achieving effective doses within tumour. To address this issue and ascertain the radiosensitisation potential of simvastatin, we developed a parenteral high density lipoprotein nanoparticle (HDL NP) formulation of this commonly used statin. A scalable method for the preparation of the simvastatin-HDL NPs was developed using a 3D printed microfluidic mixer. This enables the production of litre scale amounts of particles with minimal batch to batch variation. Simvastatin-HDL NPs enhanced the radiobiological response in 2D/3D head and neck squamous cell carcinoma (HNSCC) in vitro models. The simvastatin-HDL NPs radiosensitisation was comparable to that of 10 and 5 times higher doses of free drug in 2D and 3D cultures, respectively, which could be partially explained by more efficient cellular uptake of the statin in the nanoformulation as well as by the inherent biological activity of the HDL NPs on the cholesterol pathway. The radiosensitising potency of the simvastatin-HDL nanoformulation was validated in an immunocompetent MOC-1 HNSCC tumour bearing mouse model. This data supports the rationale of repurposing statins through reformulation within HDL NPs. Statins are safe and readily available molecules including as generic, and their use as radiosensitisers could lead to much needed effective and affordable approaches to improve treatment of solid tumours.

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.

Pharmacological Modulation of B16‐F10 Murine Melanoma‐Induced TAMs Functional Phenotype via Lipoprotein‐Based Nanoparticles

Tumor‐associated macrophages (TAMs) are one of the most abundant immune cells in the tumor microenvironment (TME). Their immunosuppressive phenotype (M2) promotes tumor progression. The re‐education of TAMs in the TME to a pro‐immune phenotype (M1) is considered to enhance the effectiveness of the anti‐tumor immune response and leads to tumor regression. An effective therapeutic approach utilizing macrophage re‐polarization will likely require the selective delivery of agents to the TME to execute the M2‐to‐M1 phenotype transformation. This strategy is known to avoid adverse immune‐related events through the utilization of nanocarriers that can effectively penetrate the TME and interact with TAMs. As a drug delivery platform, reconstituted high‐density lipoprotein nanoparticles (rHDL NPs) have attracted increased interest in cancer therapeutics, owing to their biocompatibility and their capacity to transport lipophilic drugs toward their accumulation in the tumor. We and others have shown that the scavenger receptor class B type 1 (SR‐B1), at the surface of target cells, mediates the intracellular (non‐endocytic) delivery of rHDL NPs’ payload. The novel lipophilic agonist of the STimulator of INterferon Genes (STING), MSA‐2, has been used successfully to mediate the M2‐to‐M1 reversal in murine models of cancer. In this study, we aim to deliver MSA‐2 to TAMs using mannose‐decorated rHDL NPs to induce a conversion to an M1 phenotype. Because TAMs highly express the mannose receptor CD206 that facilitates the interaction of mannose with TAMs, inducing an M1 phenotype, we anticipate a successful outcome based on this strategy. We prepared and validated the retention of the PEGylated mannose moiety on the rHDL NPs. Fluorescence anisotropy and absorbance studies showed that rHDL NPs successfully encapsulated MSA‐2. IL‐4‐induced M2 macrophages and B16‐F10 murine melanoma conditioned media‐induced TAMs showed a higher SR‐B1 expression compared to M1 macrophages. The lower uptake of the surrogate dye payload by M1 macrophages (compared to M2 macrophages and TAMs) from the mannose‐decorated rHDL NPs, corroborates the importance of the SR‐B1 receptor in rHDL payload delivery. The above findings have significant therapeutic implications for the strategy of utilizing mannose‐decorated rHDL‐NPs to selectively target TAMs in the tumor microenvironment. In addition, this formulation of MSA‐2 with the mannose‐decorated rHDL NPs can be applied in a synergistic combination with mainstream immunotherapies to improve treatment outcomes for cancer patients, diagnosed with solid tumors.

ApoA-I Nanoparticles as Curcumin Carriers for Cerebral Endothelial Cells: Improved Cytoprotective Effects against Methylglyoxal

Methylglyoxal (MGO) is a highly reactive metabolite of glucose present at elevated levels in diabetic patients. Its cytotoxicity is associated with endothelial dysfunction, which plays a role in cardiovascular and cerebrovascular complications. Although curcumin has many therapeutic benefits, these are limited due to its low bioavailability. We aimed to improve the bioavailability of curcumin and evaluate a potential synergistic effect of curcumin and reconstituted high-density lipoprotein (rHDL) nanoparticles (Cur-rHDLs) on MGO-induced cytotoxicity and oxidative stress in murine cerebrovascular endothelial cells (bEnd.3). Cur-rHDL nanoparticles (14.02 ± 0.95 nm) prepared by ultracentrifugation and containing curcumin were quantified by LC–MS/MS. The synergistic effect of cur-rHDL nanoparticles was tested on bEnd.3 cytotoxicity, reactive oxygen species (ROS) production, chromatin condensation, endoplasmic reticulum (ER) stress, and endothelial barrier integrity by impedancemetry. The uptake of curcumin, alone or associated with HDLs, was also assessed by mass spectrometry. Pretreatment with Cur-rHDLs followed by incubation with MGO showed a protective effect on MGO-induced cytotoxicity and chromatin condensation, as well as a strong protective effect on ROS production, endothelial cell barrier integrity, and ER stress. These results suggest that Cur-rHDLs could be used as a potential therapeutic agent to limit MGO-induced dysfunction in cerebrovascular endothelial cells by enhancing the bioavailability and protective effects of curcumin.

High density lipoprotein nanoparticle as delivery system for radio-sensitising miRNA: An investigation in 2D/3D head and neck cancer models

Radiotherapy is one of the main treatment options for head and neck cancer patients. However, its clinical efficacy is hindered by both radiation induced side effects and radio-resistance. Radio-sensitising approaches with acceptable toxicity are being actively investigated. Among these, RNA therapeutics have great potentials as radio-sensitisers owing to their ability to target pathways specific to radio-resistance. However, their clinical translation is challenging due to delivery issues. Herein, we report the application of high-density lipoprotein nanoparticle (HDL NPs) as a biocompatible delivery system for a well-established radio-sensitising RNA, miR-34a. A simple/fast microfluidic based technique was used to prepare miR-34a-HDL NPs. Profiling of the radiation response in the UM-SCC-1 head and neck cancer cell line confirmed reduced metabolic activity and increased radiation induced apoptosis upon treatment with miR-34a-HDL NPs. The radio-sensitising properties of miR-34a-HDL NPs were further confirmed in a more biologically relevant co-culture spheroid model of head and neck cancer. Increased apoptotic activity and disrupted cell cycle were induced by miR-34a delivered by HDL NPs. The enhanced radio-biologic effects observed in both 2D and 3D models confirmed the utility of HDL NPs as an efficient delivery system for radio-sensitising RNA.

Targeted photodynamic therapy using reconstituted high-density lipoproteins as rhodamine transporters

Reconstituted high-density lipoprotein (rHDL) nanoparticles are excellent transporters of molecules and very useful for targeted therapy as they specifically recognize the scavenger receptor, class B1 (SR-B1) that is present on the surface of a wide range of tumor cells. However, they have rarely been employed to transport photosensitizers (PS) for photodynamic therapy (PDT). Rhodamine (R) compounds have been dismissed as useful PSs for PDT due to their low 1O2 production, excitation wavelengths with little tissue penetration, and poor selectivity for tumor cells. It was recently demonstrated that when irradiating at 532 nm or with Cerenkov radiation (CR) from a β-emitting radionuclide, R123, R6G, and RB undergo electron transfer reactions (type I reaction) with folic acid. R6G also produces type I reactions with O2. In this work, the photodynamic effects of the rHDL-R system were evaluated in vitro. rHDL nanoparticles loaded with R123, R6G, and RB were synthesized, and the PS was internalized into T47D tumor cells. When cells were irradiated with a 532-nm laser in the presence of an rHDL-R systems, a cytotoxic photodynamic effect was obtained in the order R6G > R123 > RB. In the presence of CR from a 177Lu source, cytotoxicity showed the order R6G > RB > R123. The higher cytotoxicity induced by R6G in both cases corresponds to higher cellular internalization and larger production of type I and II reactions. Thus, in this work, it is proposed that rHDL-R/177Lu system can be applied in theragnostics as a multimodal radiotherapy-PDT-imaging system (imaging by SPECT or Cerenkov) and in hypoxic solid tumors in which external radiation is not effective and 177Lu-CR acts as light source.

Synthesis and Characterization of Salinomycin-Loaded High-Density Lipoprotein and Its Effects on Cervical Cancer Cells and Cervical Cancer Stem Cells.

BackgroundCervical cancer stem cells (CCSCs), a small part of tumor population, are one of the important reasons for metastasis and recurrence of cervical cancer. Targeting CCSCs may be an effective way to eliminate tumors. Salinomycin (Sal) has been proved to be an effective anticancer drug in many studies, especially for cancer stem cells (CSCs). However, the cytotoxicity of salinomycin limits its further research as an anticancer drug. High-density lipoprotein (HDL) nanoparticles are an excellent drug carrier, which can reduce the toxicity of Sal, have a certain targeting effect and improve the clinical benefit of Sal.MethodsSalinomycin-loaded high-density lipoprotein (S-HDL) was synthesized and characterized by various analytical techniques. CD44highCD24low CCSCs were isolated from HeLa cells by magnetic separation. The uptake of HDL nanoparticles was observed by laser confocal microscopy, and the effect of S-HDL on the proliferation of CCCs and CCSCs was detected by cell viability analysis. Genome-wide analysis was used to analyze the effects of S-HDL on the biological processes of CCCs and then cell apoptosis, cell cycle and cell migration were selected for verification.ResultsS-HDL had a particle size of 38.98 ± 1.78 nm and an encapsulation efficiency of 50.73 ± 4.29%. Cell uptake analysis showed that HDL nanoparticles could enhance the drug uptake of CCCs and CCSCs and may target CCCs and CCSCs. In cell viability analysis, CCCs and CCSCs showed high sensitivity to S-HDL. S-HDL can more efficiently prevent CCSCs from developing tumorspheres than Sal in tumorsphere formation study. S-HDL had stronger ability to induce cell cycle arrest, promote cell apoptosis and inhibit cell migration compared with free Sal, which was consistent with the results of Genome Wide analysis.ConclusionS-HDL can effectively target and eliminate CCCs and CCSCs, which is a potential drug for the treatment of cervical cancer.

Peptide-Targeted High-Density Lipoprotein Nanoparticles for Combinatorial Treatment against Metastatic Breast Cancer.

The sonic hedgehog (SHH) signaling pathway exhibits aberrant activation in triple-negative breast cancer (TNBC), wherein it regulates several malignant phenotypes related to tumor metastasis. GANT61, an inhibitor of the SHH signaling pathway, may offer promise when administered in combination with conventional chemotherapy to treat metastatic TNBC. However, poor bioavailability and substantial off-target toxicity limit its clinical application. To address these limitations, we designed a peptide-functionalized dual-targeting delivery system encapsulating paclitaxel and GANT61 in tLyP-1 peptide-modified reconstituted high-density lipoprotein nanoparticle (tLyP-1-rHDL-PTX/GANT61 NP) for metastatic TNBC treatment. The apolipoprotein A-1 and tLyP-1 peptide modified on the surface of nanoparticles enable the delivery system to target tumor cells by binding to the overexpressed scavenger receptor B type I and neuropilin-1 receptor. Moreover, the tLyP-1 peptide also enables the deep tumor penetration of nanoparticles further facilitating paclitaxel and GANT61 delivery. Increased cellular uptake of the nanoparticles was observed in both MDA-MB-231, BT-549 tumor cells, and their 3D tumor spheroids. A series of in vitro experiments reveal that GANT61 was able to suppress key metastasis-related tumor cell activities including angiogenesis, migration, invasion, and stemness. Owing to more effective drug administration, the metastasis suppression efficiency of GANT61 was significantly enhanced by the dual-targeting tLyP-1-rHDL delivery system. Meanwhile, the codelivery of paclitaxel and GANT61 by dual-targeting tLyP-1-rHDL nanoparticles demonstrated superior efficiency of disrupting proliferation and inducing apoptosis in tumor cells compared with drug solutions. In a spontaneous metastasis breast cancer NCG mice model, the tLyP-1-rHDL-PTX/GANT61 nanoparticles exhibited highly tumor-specific distribution and result in significant inhibition of the primary tumor growth and dramatic reduction of lung metastasis without obvious side effects. The present work suggests that a combination of the SHH signaling pathway suppression and chemotherapy assisted by peptide-functionalized targeting tLyP-1-rHDL nanoparticles may provide a promising strategy for metastatic TNBC treatment.

(Invited) Optical Control of the Membrane Potential of Living Cells with Donor-Acceptor-Linked Molecules

In living animal cells, there exists a difference in the electrical charge on the two extreme sides of the plasma membrane, that is, a slight excess of positive and negative ions outside and inside a cell, respectively. Such a charge difference produces a membrane potential (V m) across the plasma membrane. The V m (< 0) is controlled by various ion channels, and, especially, in neuronal cells, it is drastically changed for neurotransmission. In response to some input, the ion channels of neuronal cells get quickly activated such that the abovementioned difference is cancelled (∆V m > 0, depolarization). This electrical signal propagates along the neuronal axons like a wave through coordinated activation and deactivation of the ion channels. Therefore, the control of membrane potential and ion transport across the plasma membrane using a light source is an attractive strategy that can allow targeted, fast control of precisely defined events in the plasma membrane. In this symposium, we have demonstrated a novel general strategy for the control of membrane potential and ion transport by using donor-acceptor-linked molecules (D-A molecules) capable of intramolecular long-lived charge separation, nanocarriers for solubilization, membrane targeting of D-A molecules, and light (Figure 1).For the preparation of the D-A molecule, porphyrin (H2P) derivatives with ferrocene (Fc) and fullerene (C60) were synthesized to create a triadic arrangement (Fc-H2P-C60). We had previously reported that, under light irradiation, Fc-H2P-C60 yields the ferrocenium cation (Fc+)-H2P-C60 radical anion (C60 •-) with a long lifetime of ~0.01 ms [1]. For its solubilization in the physiological media and safer plasma membrane delivery, we focused on a lipoprotein in our body considering its amphiphilic property. High-density lipoprotein (HDL) can be reconstituted with recombinant lipid-binding proteins and phospholipids. We had previously reported that HDL genetically and chemically engineered with cytophilic peptides and fusogenic lipids is capable of high plasma membrane binding without significant cytotoxicity [2]. For this purpose, Fc-H2P-C60 was solubilized with one of our engineered HDL nanoparticles (cpHDL) and added to a cell culture medium of rat pheochromocytoma cells (PC12). The cell membrane delivery of Fc-H2P-C60 was suggested by confocal analysis and freeze-fracture electron microscopy. Subsequent light irradiation of PC12 cells increased the membrane potential (depolarization), which was not observed for the reference molecule (H2P) that was incapable of charge separation [3]. In addition, the inhibition of potassium ion flow across the membrane was found to be responsible for this depolarization (Figure 1). Our following study with 3 D-A molecules, including Fc-H2P-C60, revealed that the degree of the depolarization was positively correlated with the charge separation yield [4]. In this symposium, I will discuss more about the potential of applying our D-A molecules/carriers system in the biomedical fields.Figure 1. Schematic illustration of optical control of membrane potential and cation flow using a D-A molecule (Fc-H2P-C60)[1] Imahori, H. et al., J. Am. Chem. Soc. 2001, 123, 2607-2617.[2] Murakami, T., Biotechncol. J. 2012, 7, 762-767.; Kim, H. et al. Biochim. Biophys. Acta Biomembr. 2019, 1861, 183008.[3] Numata, T. et al., J. Am. Chem. Soc. 2012, 134, 6092-6095.[4] Takano, Y. et al., Chem. Sci. 2016, 7, 3331-3337. Figure 1