Fredrik Melander

Synthesis and Biophysical Characterization of Chlorambucil Anticancer Ether Lipid Prodrugs

The synthesis and biophysical characterization of four prodrug ether phospholipid conjugates are described. The lipids are prepared from the anticancer drug chlorambucil and have C16 and C18 ether chains with phosphatidylcholine or phosphatidylglycerol headgroups. All four prodrugs have the ability to form unilamellar liposomes (86-125 nm) and are hydrolyzed by phospholipase A(2), resulting in chlorambucil release. Liposomal formulations of prodrug lipids displayed cytotoxicity toward HT-29, MT-3, and ES-2 cancer cell lines in the presence of phospholipase A(2), with IC(50) values in the 8-36 microM range.

Mechanistic study of the sPLA2-mediated hydrolysis of a thio-ester pro anticancer ether lipid.

Secretory phospholipase A(2) (sPLA(2)) is an interesting enzyme for triggered liposomal drug delivery to tumor tissue due the overexpression of sPLA(2) in cancerous tissue. A drug delivery system based on the triggered release of therapeutics from sPLA(2)-sensitive liposomes constituted of pro anticancer ether lipids, which become cytotoxic upon sPLA(2)-catalyzed hydrolysis has previously been established. To optimize the hydrolysis rate of the lipids and thereby optimizing the release profile of the drugs from the liposomes, we have synthesized a thio-ester pro anticancer ether lipid. Liposomes constituted of this lipid showed an altered rate of hydrolysis by sPLA(2). We have tested the cytotoxicity of the thio-ester pro anticancer ether lipids toward cancer cells, and the results showed that the cytotoxicity is indeed maintained upon sPLA(2) exposure. To further understand the origin for the observed different hydrolysis rates for the esters, we have applied molecular dynamics simulations and density functional theory. The combination of these theoretical methods has given valuable insight into the molecular mechanism for sPLA(2) action on sulfur-containing phospholipids. It appears that the enzyme-catalyzed hydrolysis of thio-esters follow a different pathway compared to the hydrolysis pathway of the free thio-ester.

Impedimetric Toxicity Assay in Microfluidics Using Free and Liposome-Encapsulated Anticancer Drugs

In this work, we have developed a microfluidic cytotoxicity assay for a cell culture and detection platform, which enables both fluid handling and electrochemical/optical detection. The cytotoxic effect of anticancer drugs doxorubicin (DOX), oxaliplatin (OX) as well as OX-loaded liposomes, developed for targeted drug delivery, was evaluated using real-time impedance monitoring. The time-dependent effect of DOX on HeLa cells was monitored and found to have a delayed onset of cytotoxicity in microfluidics compared with static culture conditions based on data obtained in our previous study. The result of a fluorescent microscopic annexin V/propidium iodide assay, performed in microfluidics, confirmed the outcome of the real-time impedance assay. In addition, the response of HeLa cells to OX-induced cytotoxicity proved to be slower than toxicity induced by DOX. A difference in the time-dependent cytotoxic response of fibrosarcoma cells (HT1080) to free OX and OX-loaded liposomes was observed and attributed to incomplete degradation of the liposomes, which results in lower drug availability. The matrix metalloproteinase (MMP)-dependent release of OX from OX-loaded liposomes was also confirmed using laryngopharynx carcinoma cells (FaDu). The comparison and the observed differences between the cytotoxic effects under microfluidic and static conditions highlight the importance of comparative studies as basis for implementation of microfluidic cytotoxic assays.

Targeting transferrin receptors at the blood-brain barrier improves the uptake of immunoliposomes and subsequent cargo transport into the brain parenchyma

Drug delivery to the brain is hampered by the presence of the blood-brain barrier, which excludes most molecules from freely diffusing into the brain, and tightly regulates the active transport mechanisms that ensure sufficient delivery of nutrients to the brain parenchyma. Harnessing the possibility of delivering neuroactive drugs by way of receptors already present on the brain endothelium has been of interest for many years. The transferrin receptor is of special interest since its expression is limited to the endothelium of the brain as opposed to peripheral endothelium. Here, we investigate the possibility of delivering immunoliposomes and their encapsulated cargo to the brain via targeting of the transferrin receptor. We find that transferrin receptor-targeting increases the association between the immunoliposomes and primary endothelial cells in vitro, but that this does not correlate with increased cargo transcytosis. Furthermore, we show that the transferrin receptor-targeted immunoliposomes accumulate along the microvessels of the brains of rats, but find no evidence for transcytosis of the immunoliposome. Conversely, the increased accumulation correlated both with increased cargo uptake in the brain endothelium and subsequent cargo transport into the brain. These findings suggest that transferrin receptor-targeting is a relevant strategy of increasing drug exposure to the brain.

18F-FDG PET/CT-based early treatment response evaluation of nanoparticle-assisted photothermal cancer therapy

Within the field of nanoparticle-assisted photothermal cancer therapy, focus has mostly been on developing novel heat-generating nanoparticles with the right optical and dimensional properties. Comparison and evaluation of their performance in tumor-bearing animals are commonly assessed by changes in tumor volume; however, this is usually a late-occurring event. This study implements 2-deoxy-2-[F-18]fluoro-D-glucose positron emission tomography imaging to perform early evaluation of the treatment outcome of photothermal therapy. Silica-gold nanoshells (NS) are administered intravenously to nude mice bearing human neuroendocrine tumor xenografts and the tumors are irradiated by a near-infrared laser. The animals are positron emission tomography scanned with 2-deoxy-2-[F-18]fluoro-D-glucose one day before and one day after treatment. Using this setup, a significant decrease in tumor uptake of 2-deoxy-2-[F-18]fluoro-D-glucose is found already one day after therapy in the group receiving NS and laser treatment compared to control animals. At this time point no change in tumor volume can be detected. Moreover, the change in tumor uptake, is used to stratify the animals into responders and non-responders, where the responding group matched improved survival. Overall, these findings support the use of 2-deoxy-2-[F-18]fluoro-D-glucose positron emission tomography imaging for preclinical and clinical evaluation and optimization of photothermal therapy.

Synthesis of tocopheryl succinate phospholipid conjugates and monitoring of phospholipase A2 activity

Tocopheryl succinates (TOSs) are, in contrast to tocopherols, highly cytotoxic against many cancer cells. In this study the enzyme activity of secretory phospholipase A(2) towards various succinate-phospholipid conjugates has been investigated. The synthesis of six novel phospholipids is described, including two TOS phospholipids conjugates. The studies revealed that the TOS conjugates are poor substrates for the enzyme whereas the phospholipids with alkyl and phenyl succinate moieties were hydrolyzed by the enzyme to a high extent.

Antibody affinity and valency impact brain uptake of transferrin receptor-targeted gold nanoparticles

Rationale: The ability to treat invalidating neurological diseases is impeded by the presence of the blood-brain barrier (BBB), which inhibits the transport of most blood-borne substances into the brain parenchyma. Targeting the transferrin receptor (TfR) on the surface of brain capillaries has been a popular strategy to give a preferential accumulation of drugs or nanomedicines, but several aspects of this targeting strategy remain elusive. Here we report that TfR-targeted gold nanoparticles (AuNPs) can accumulate in brain capillaries and further transport across the BBB to enter the brain parenchyma. Methods: We characterized our targeting strategy both in vitro using primary models of the BBB and in vivo using quantitative measurements of gold accumulation by inductively-coupled plasma-mass spectrometry together with morphological assessments using light microscopy after silver enhancement and transmission electron microscopy with energy-dispersive X-ray spectroscopy. Results: We find that the uptake capacity is significantly modulated by the affinity and valency of the AuNP-conjugated antibodies. Specifically, antibodies with high and low affinities mediate a low and intermediate uptake of AuNPs into the brain, respectively, whereas a monovalent (bi-specific) antibody improves the uptake capacity remarkably. Conclusion: Our findings indicate that monovalent ligands may be beneficial for obtaining transcytosis of TfR-targeted nanomedicines across the BBB, which is relevant for future design of nanomedicines for brain drug delivery.

Multiplexed Dosing Assays by Digitally Definable Hydrogel Volumes.

Stable and low-cost multiplexed drug sensitivity assays using small volumes of cells or tissue are in demand for personalized medicine, including patient-specific combination chemotherapy. Spatially defined projected light photopolymerization of hydrogels with embedded active compounds is introduced as a flexible and cost-efficient method for producing multiplexed dosing assays. The high spatial resolution of light projector technology defines multiple compound doses by the volume of individual compound-embedded hydrogel segments. Quantitative dosing of multiple proteins with a dynamic range of 1-2 orders of magnitude is demonstrated using fluorescently labeled albumins. The hydrogel matrix results from photopolymerization of low-cost poly(ethylene glycol) diacrylates (PEGDA), and tuning of the PEGDA composition enables fast complete dosing of all tested species. Dosing of hydrophilic and hydrophobic compounds is demonstrated using two first-line chemotherapy regimens combining oxaliplatin, SN-38, 5-fluorouracil, and folinic acid, with each compound being dosed from a separate light-defined hydrogel segment. Cytotoxicity studies using a colorectal cancer cell line show equivalent effects of dissolved and released compounds. Further control of the dosing process is demonstrated by liposomal encapsulation of oxaliplatin, stable embedding of the liposomes in hydrogels for more than 3 months, and heat-triggered complete release of the loaded oxaliplatin.

Endothelial Protein C–Targeting Liposomes Show Enhanced Uptake and Improved Therapeutic Efficacy in Human Retinal Endothelial Cells

Purpose To determine whether human retinal endothelial cells (HRECs) express the endothelial cell protein C receptor (EPCR) and to realize its potential as a targeting moiety by developing novel single and dual corticosteroid-loaded functionalized liposomes that exhibit both enhanced uptake by HRECs and superior biologic activity compared to nontargeting liposomes and free drug. Methods EPCR expression of HRECs was investigated through flow cytometry and Western blot assays. EPCR-targeting liposomes were developed by functionalizing EPCR-specific antibodies onto liposomes, and the uptake of liposomes was assessed with flow cytometry and confocal laser scanning microscopy. The therapeutic potential of EPCR-targeting liposomes was determined by loading them with prednisolone either through bilayer insertion and/or by remote loading into the aqueous core. The carrier efficacy was assessed in two ways through its ability to inhibit secretion of interleukins in cells stimulated with high glucose and angiogenesis in vitro by using an endothelial cell tube formation assay. Results HRECs express EPCR at a similar level in both human aortic and umbilic vein endothelial cells. The EPCR-targeting liposomes displayed at least a 3-fold higher uptake compared to nontargeting liposomes. This enhanced uptake was translated into superior anti-inflammatory efficacy, as the corticosteroid-loaded EPCR-targeting liposomes significantly reduced the secretion of IL-8 and IL-6 and inhibited the development of cell tube formations in contrast to nontargeting liposomes. Conclusions We show that HRECs express EPCR and this receptor could be a promising nanomedicine target in ocular diseases where the endothelial barrier of the retina is compromised.

Multifarious Biologic Loaded Liposomes that Stimulate the Mammalian Target of Rapamycin Signaling Pathway Show Retina Neuroprotection after Retina Damage

A common event in optic neuropathies is the loss of axons and death of retinal ganglion cells (RGCs) resulting in irreversible blindness. Mammalian target of rapamycin (mTOR) signaling pathway agonists have been shown to foster axon regeneration and RGC survival in animal models of optic nerve damage. However, many challenges remain in developing therapies that exploit cell growth and tissue remodeling including (i) activating/inhibiting cell pathways synergistically, (ii) avoiding tumorigenesis, and (iii) ensuring appropriate physiological tissue function. These challenges are further exacerbated by the need to overcome ocular physiological barriers and clearance mechanisms. Here we present liposomes loaded with multiple mTOR pathway stimulating biologics designed to enhance neuroprotection after retina damage. Liposomes were loaded with ciliary neurotrophic factor, insulin-like growth factor 1, a lipopeptide N-fragment osteopontin mimic, and lipopeptide phosphatase tension homologue inhibitors for either the ATP domain or the c-terminal tail. In a mouse model of N-methyl-d-aspartic acid induced RGC death, a single intravitreal administration of liposomes reduced both RGC death and loss of retina electrophysiological function. Furthermore, combining liposomes with transplantation of induced pluripotent stem cell derived RGCs led to an improved electrophysiological outcome in mice. The results presented here show that liposomes carrying multiple signaling pathway modulators can facilitate neuroprotection and transplant electrophysiological outcome.