Monitoring cell endocytosis of liposomes by real-time electrical impedance spectroscopy.

Evaluation and understanding the effect of drug delivery in in vitro systems is fundamental in drug discovery. We present an assay based on real-time electrical impedance spectroscopy (EIS) measurements that can be used to follow the internalisation and cytotoxic effect of a matrix metalloproteinase (MMP)-sensitive liposome formulation loaded with oxaliplatin (OxPt) on colorectal cancer cells. The EIS response identified two different cellular processes: (i) a negative peak in the cell index (CI) within the first 5 h, due to onset of liposome endocytosis, followed by (ii) a subsequent CI increase, due to the reattachment of cells until the onset of cytotoxicity with a decrease in CI. Free OxPt or OxPt-loaded Stealth liposomes did not show this two-stage EIS response; the latter can be due to the fact that Stealth cannot be cleaved by MMPs and thus is not taken up by the cells. Real-time bright-field imaging supported the EIS data, showing variations in cell adherence and cell morphology after exposure to the different liposome formulations. A drastic decrease in cell coverage as well as rounding up of cells during the first 5 h of exposure to OxPt-loaded (MMP)-sensitive liposome formulation is reflected by the first negative EIS response, which indicates the onset of liposome endocytosis. Graphical abstract.

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.

HER2-targeted, enzyme-activated liposomes show superior in vivo efficacy in an ovarian cancer model.

Liposomes carrying chemotherapeutic drugs can accumulate passively in solid tumors at high levels. However, additional targeting of the liposomes towards e.g. receptors expressed on cancer cells may improve their interaction and therapeutic properties. In this study, we designed a liposomal delivery system, which utilizes the intrinsic characteristics of HER2-positive tumors to ensure efficient delivery of oxaliplatin to the cancer cells. On the liposome surface, trastuzumab, an antibody specific to the HER2 receptor, was shown to facilitate internalization by the cancer cells. A polyethylene glycol (PEG) layer on the liposome surface provides protection from mononuclear phagocyte system uptake. To optimize the interaction between liposomes and cancer cells, a protease-sensitive cleavable peptide linker was inserted at the base of each PEG. The PEG layer is then cleaved off by intra- and extracellular matrix metalloproteinases (MMPs) upon accumulation in the tumor. Our data demonstrate that the removal of PEG significantly destabilizes the liposomes and leads to substantial oxaliplatin release. The proposed beneficial effect of combining antibody-mediated internalization with MMP sensitivity was confirmed in a series of in vivo studies using ovarian cancer xenograft models. The results demonstrated that HER2-targeted MMP-sensitive liposomes have superior anticancer activity compared to non-targeted and non-cleavable liposomes.

Active Transport and Ocular Distribution of Intravitreally Injected Liposomes.

Purpose: Drug delivery to the retina remains a challenge due to ocular barriers and fast clearing mechanisms. Nanocarrier drug delivery systems (NDDSs) hold the promise of prolonging intraocular retention times and increasing drug concentrations in the retina. Methods: Anionic and cationic PEGylated liposomes, loaded with oxaliplatin (OxPt) to be used as trace element, were prepared from dry lipid powders. The differently charged liposomes were intravitreally injected in C57BL/6JrJ mice; eyes were harvested 2 hours and 24 hours post-injection. To investigate active transport mechanisms in the eye, a subset of mice were pre-injected with chloroquine before injection with cationic liposomes. Eyes were dissected and the distribution of OxPt in different tissues were quantified by inductively coupled plasma mass spectrometry (ICP-MS). Results: Both liposome formulations enhanced the retention time of OxPt in the vitreous over free OxPt. Surprisingly, when formulated in cationic liposomes, OxPt translocated through the retina and accumulated in the RPE-sclera. Pre-injection with chloroquine inhibited the transport of liposomal OxPt from the vitreous to the RPE-sclera. Conclusions: We show that liposomes can enhance the retention time of small molecular drugs in the vitreous and that active transport mechanisms are involved in the trans retinal transport of NDDS after intravitreal injections. Translational Relevance: These results highlight the need for understanding the dynamics of ocular transport mechanisms in living eyes when designing NDDS with the back of the eye as the target. Active transport of nanocarriers through the retina will limit the drug concentration in the neuronal retina but might be exploited for targeting the RPE.

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.

Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks.

We show that DNA double-strand breaks (DSBs) induce complex subcompartmentalization of genome surveillance regulators. Chromatin marked by gamma-H2AX is occupied by ataxia telangiectasia-mutated (ATM) kinase, Mdc1, and 53BP1. In contrast, repair factors (Rad51, Rad52, BRCA2, and FANCD2), ATM and Rad-3-related (ATR) cascade (ATR, ATR interacting protein, and replication protein A), and the DNA clamp (Rad17 and -9) accumulate in subchromatin microcompartments delineated by single-stranded DNA (ssDNA). BRCA1 and the Mre11-Rad50-Nbs1 complex interact with both of these compartments. Importantly, some core DSB regulators do not form cytologically discernible foci. These are further subclassified to proteins that connect DSBs with the rest of the nucleus (Chk1 and -2), that assemble at unprocessed DSBs (DNA-PK/Ku70), and that exist on chromatin as preassembled complexes but become locally modified after DNA damage (Smc1/Smc3). Finally, checkpoint effectors such as p53 and Cdc25A do not accumulate at DSBs at all. We propose that subclassification of DSB regulators according to their residence sites provides a useful framework for understanding their involvement in diverse processes of genome surveillance.

Photothermal Therapy as Adjuvant to Surgery in an Orthotopic Mouse Model of Human Fibrosarcoma.

Surgery is still the first-line treatment for multiple solid cancers. However, recurrence is a common issue, especially when dealing with aggressive tumors or tumors that are difficult to completely remove due to their location. Getting clear surgical margins can be challenging, but treatment strategies combining surgery with other anti-cancer therapies can potentially improve the outcome. Photothermal therapy (PTT) is a technique that relies on photoabsorbing agents, such as gold nanoparticles, to transform light into local hyperthermia. This technique can be used to ablate tumor tissue where the photoabsorbing agent accumulates, sparing healthy surrounding tissue. In this study, we examined the potential of gold nanoparticle-based PTT as an adjuvant treatment to surgery in a mouse model of human fibrosarcoma. For this we performed subtotal tumor resection to mimic a clinical situation where total tumor removal is not achieved, and subsequent PTT was applied on the surgical field. Our results showed that animals undergoing adjuvant PTT after surgery presented sustained delayed tumor growth and improved survival when compared to animals that only underwent surgery. We believe that these findings show the potential of PTT as an adjuvant method to traditional tumor surgery and could pave way to more personalized treatment options.

Effective Intratumoral Retention of [103 Pd]AuPd Alloy Nanoparticles Embedded in Gel-Forming Liquids Paves the Way for New Nanobrachytherapy.

Local application of radioactive sources as brachytherapy is well established in oncology. This treatment is highly invasive however, due to the insertion of millimeter sized metal seeds. The authors report the development of a new concept for brachytherapy, based on gold-palladium (AuPd) alloy nanoparticles, intrinsically radiolabeled with 103 Pd. These are formulated in a carbohydrate-ester based liquid, capable of forming biodegradable gel-like implants upon injection. This allows for less invasive administration through small-gauge needles. [103 Pd]AuPd nanoparticles with sizes around 20 nm are prepared with radiolabeling efficiencies ranging from 79% to >99%. Coating with the hydrophobic polymer poly(N-isopropylacrylamide) leads to nanoparticle diameters below 40 nm. Dispersing the nanoparticles in ethanol with water insoluble carbohydrate esters gives "nanogels", a low viscosity liquid capable of solidifying upon injection into aqueous environments. Both nanoparticles and radioactivity are stably retained in the nanogel over 25 days (>99%) after formation in aqueous buffers. Animals bearing CT26 murine tumors are injected intratumorally with 25 MBq of the 103 Pd-nanogel, and display tumor growth delay and significantly increase median survival times compared with control groups. Excellent retention in the tumor of both the 103 Pd and the nanoparticle matrix itself is observed, demonstrating a potential for replacing currently used brachytherapy seeds.

p38-MAPK signals survival by phosphorylation of caspase-8 and caspase-3 in human neutrophils.

Neutrophil apoptosis occurs both in the bloodstream and in the tissue and is considered essential for the resolution of an inflammatory process. Here, we show that p38-mitogen-activated protein kinase (MAPK) associates to caspase-8 and caspase-3 during neutrophil apoptosis and that p38-MAPK activity, previously shown to be a survival signal in these primary cells, correlates with the levels of caspase-8 and caspase-3 phosphorylation. In in vitro experiments, immunoprecipitated active p38-MAPK phosphorylated and inhibited the activity of the active p20 subunits of caspase-8 and caspase-3. Phosphopeptide mapping revealed that these phosphorylations occurred on serine-364 and serine-150, respectively. Introduction of mutated (S150A), but not wild-type, TAT-tagged caspase-3 into primary neutrophils made the Fas-induced apoptotic response insensitive to p38-MAPK inhibition. Consequently, p38-MAPK can directly phosphorylate and inhibit the activities of caspase-8 and caspase-3 and thereby hinder neutrophil apoptosis, and, in so doing, regulate the inflammatory response.

Dynamic assembly and sustained retention of 53BP1 at the sites of DNA damage are controlled by Mdc1/NFBD1.

53BP1 is a key component of the genome surveillance network activated by DNA double strand breaks (DSBs). Despite its known accumulation at the DSB sites, the spatiotemporal aspects of 53BP1 interaction with DSBs and the role of other DSB regulators in this process remain unclear. Here, we used real-time microscopy to study the DSB-induced redistribution of 53BP1 in living cells. We show that within minutes after DNA damage, 53BP1 becomes progressively, yet transiently, immobilized around the DSB-flanking chromatin. Quantitative imaging of single cells revealed that the assembly of 53BP1 at DSBs significantly lagged behind Mdc1/NFBD1, another DSB-interacting checkpoint mediator. Furthermore, short interfering RNA-mediated ablation of Mdc1/NFBD1 drastically impaired 53BP1 redistribution to DSBs and triggered premature dissociation of 53BP1 from these regions. Collectively, these in vivo measurements identify Mdc1/NFBD1 as a key upstream determinant of 53BP1's interaction with DSBs from its dynamic assembly at the DSB sites through sustained retention within the DSB-flanking chromatin up to the recovery from the checkpoint.

Preclinical evaluation of cationic DOTA-triarginine-lipid conjugates for theranostic liquid brachytherapy.

Liquid brachytherapy is an emerging technology for internal radiation therapy where liquids containing radionuclides are administered directly into solid tumors. These technologies are less invasive than conventional brachytherapy, and can potentially improve the dose coverage and homogeneity of the radioactivity distribution within the tumor. For this purpose, we have developed a novel cationic micelle system for delivery of a range of radionuclides. The system is applicable for emitters of alpha, beta or photon radiation, and enables dose-mapping via theranostic nuclear imaging. Methods: The cationic micelles were developed as linear surfactants comprising the chelator DOTA, a triarginine sequence and a palmitoyl or stearoyl fatty acid chain. The critical micelle concentration of the surfactants was determined, and the micelles were radiolabelled with 64Cu or 177Lu in high radiochemical purity (>95%). The tumor retention and biodistribution of the 64Cu-radiolabeled surfactants, administered as micelles or formulated in liposomes, were investigated in vivo by PET/CT in a tumor bearing mouse model. Results: The interaction of the micelles with anionic lipid membranes was demonstrated to be favourable, using a liposome partition assay. In vivo, the surfactants formulated both as cationic micelles and liposomes displayed the best intratumoral retention, with micelles providing more homogeneous activity distribution. Conclusion: A cationic, surfactant-based drug delivery system was developed and demonstrated promise as a vehicle for liquid brachytherapy when formulated as micelles or in liposomes. The system enables accurate dosimetry due to the flexible radiochemistry of DOTA.

Elucidating the anomalous membrane permeability of Ag(I), Cu(II), Zn(II) and Au(III) towards new nanoreactor strategies for synthesizing metal nanoparticles.

The main structural element defining the cell is the lipid membrane, which is an integral part of regulating the fluxes of ion and nutrition molecules in and out of the cell. Surprisingly, copper ions were found to have anomalous membrane permeability. This led us to consider a broader spectrum of cations and further a new approach for using liposomes as nanoreactors for synthesis of metal and metal alloy nanoparticles. In the present study, the high membrane permeability of Cu2+ and its neighbouring transition elements in the periodic table was investigated. The permeability of Ni2+, Cu2+, Zn2+, Ag+, Au3+, Mg2+, Ca2+ and Lu3+ was assessed, and we report that Zn2+, Cu2+, Ag+ and Au3+ surprisingly are able to cross lipid bilayers. This knowledge is highly relevant for understanding trafficking of cations in biological systems, as well as for design of novel nanoparticle and nanoreactor systems. An example of its use is presented as a platform for synthesizing single highly uniform gold nanoparticles inside liposomal nanoreactors. We envision that this approach could provide a new nanoreactor methodology for forming highly structurally constrained uniform metal and metal alloy nanoparticles, as well as new methods for in vivo tracking of liposomes.

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.

Modulating the antibody density changes the uptake and transport at the blood-brain barrier of both transferrin receptor-targeted gold nanoparticles and liposomal cargo.

Transport of the majority of therapeutic molecules to the brain is precluded by the presence of the blood-brain barrier (BBB) rendering efficient treatment of many neurological disorders impossible. This BBB, nonetheless, may be circumvented by targeting receptors and transport proteins expressed on the luminal surface of the brain capillary endothelial cells (BCECs). The transferrin receptor (TfR) has remained a popular target since its original description for this purpose, although clinical progression of TfR-targeted drug constructs or nanomedicines remains unsuccessful. One proposed issue pertaining to the use of TfR-targeting in nanomedicines is the efficient tuning of the ligand density on the nanoparticle surface. We studied the impact of TfR antibody density on the uptake and transport of nanoparticles into the brain, taking a parallel approach to investigate the impact on both antibody-functionalized gold nanoparticles (AuNPs) and cargo-loaded liposomes. We report that among three different low-range mean ligand densities (0.15, 0.3, and 0.6 ∗ 103 antibodies/µm2), the highest density yielded the highest ability towards both targeting of the BCECs and subsequent transport across the BBB in vivo, and in vitro using primary cultures of the murine BBB. We also find that TfR-targeting on liposomes in the mouse may induce severe adverse effects after intravenous administration.

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.

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.

Theranostic Imaging May Vaccinate against the Therapeutic Benefit of Long Circulating PEGylated Liposomes and Change Cargo Pharmacokinetics.

The enhanced permeability and retention (EPR) effect increases tumor accumulation of liposomal chemotherapy and should, in theory, increase anticancer effects and lower toxicity. Unfortunately, liposomal chemotherapy has generally not met the expected potential, perhaps because the EPR effect is not ubiquitous. PET imaging using radiolabeled liposomes can identify cancers positive for the EPR effect. In the current study, we show in clinical canine cancer patients that repeated imaging with radiolabeled liposomes (64Cu-liposome) induces the accelerated blood clearance (ABC) phenomenon. This was observed even with very long intervals between PEGylated liposome injections, which contradict previous reporting in experimental animal models. The induction of ABC may be devastating for the theranostic use of liposomal imaging, as this could vaccinate patients against therapeutic efficacy. To investigate and solve this important problem, an additional study part was designed in which rats were subjected to repeated liposomal administrations, including stealth 64Cu-liposome PET imaging and Caelyx chemotherapy. Most importantly, it was found that, by increasing the lipid dose at the first injection or by supplying a small predose before the second 64Cu-liposome injection, ABC could be prevented. Importantly, signs of liposome tracer breakdown with subsequent renal excretion were observed. These findings highlight the importance of the ABC phenomenon for liposomal predictive imaging in a clinically relevant setting and show that carefully planned application is central to avoid potential detrimental effects on patient benefit.

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.