Jeunghwan Choi

Review of biomaterial thermal property measurements in the cryogenic regime and their use for prediction of equilibrium and non-equilibrium freezing applications in cryobiology.

It is well accepted in cryobiology that the temperature history and cooling rates experienced in biomaterials during freezing procedures correlate strongly with biological outcome. Therefore, heat transfer measurement and prediction in the cryogenic regime is central to the field. Although direct measurement of temperature history (i.e. heat transfer) can be performed, accuracy is usually achieved only for local measurements within a given system and cannot be readily generalized to another system without the aid of predictive models. The accuracy of these models rely upon thermal properties which are known to be highly dependent on temperature, and in the case of significant cryoprotectant loading, also on crystallized fraction. In this work, we review the available thermal properties of biomaterials in the cryogenic regime. The review shows a lack of properties for many biomaterials in the subzero temperature domain, and especially for systems with cryoprotective agents. Unfortunately, use of values from the limited data available (usually only down to -40 degrees C) lead to an underestimation of thermal property change (i.e. conductivity rise and specific heat drop due to ice crystallization) with lower temperatures. Conversely, use of surrogate values based solely on ice thermal properties lead to an overestimation of thermal property change for most biomaterials. Additionally, recent work extending the range of available thermal properties to -150 degrees C has shown that the thermal conductivity will drop in both PBS and tissue (liver) due to amorphous/glassy phases (versus crystalline) of biomaterials with the addition of cryoprotective additives such as glycerol. Thus, we investigated the implications of using approximated or constant property values versus measured temperature-dependent values for predicting temperature history during freezing in PBS (phosphate-buffered saline) and porcine liver with and without cryoprotectants (glycerol). Using measured property values (thermal conductivity, specific heat, and latent heat of phase change) of porcine liver, a standard was created which showed that values based on surrogate ice properties under-predicted cooling times, while constant properties (i.e. based on limited data reported near the freezing point) over-predicted cooling times. Additionally, a new iterative numerical method that accommodates non-equilibrium cooling effects as a function of time and position (i.e. crystallization versus amorphous phase) was used to predict temperature history during freezing in glycerol loaded systems. Results indicate that in addition to the increase in cooling times due to the lowering of thermal diffusivity with more glycerol, non-equilibrium effects such as the prevention of maximal crystallization (i.e. amorphous phases) will further increase required cooling times. It was also found that the amplified effect of non-equilibrium cooling and crystallization with system size prevents the thermal history to be described with non-dimensional lengths, such as was possible under equilibrium cooling. These results affirm the need to use accurate thermal properties that incorporate temperature dependence and crystallized fraction. Further studies are needed to extract thermal properties of other important biomaterials in the subzero temperature domain and to develop accurate numerical methods which take into account non-equilibrium cooling events encountered in cryobiology when partial or total vitrification occurs.

In vivo photoacoustic lifetime imaging of tumor hypoxia in small animals

Abstract. Tumor hypoxia is an important factor in assessment of both cancer progression and cancer treatment efficacy. This has driven a substantial effort toward development of imaging modalities that can directly measure oxygen distribution and therefore hypoxia in tissue. Although several approaches to measure hypoxia exist, direct measurement of tissue oxygen through an imaging approach is still an unmet need. To address this, we present a new approach based on in vivo application of photoacoustic lifetime imaging (PALI) to map the distribution of oxygen partial pressure (pO2) in tissue. This method utilizes methylene blue, a dye widely used in clinical applications, as an oxygen-sensitive imaging agent. PALI measurement of oxygen relies upon pO2-dependent excitation lifetime of the dye. A multimodal imaging system was designed and built to achieve ultrasound (US), photoacoustic, and PALI imaging within the same system. Nude mice bearing LNCaP xenograft hindlimb tumors were used as the target tissue. Hypoxic regions were identified within the tumor in a combined US/PALI image. Finally, the statistical distributions of pO2 in tumor, normal, and control tissues were compared with measurements by a needle-mounted oxygen probe. A statistically significant drop in mean pO2 was consistently detected by both methods in tumors.

Nanoparticle delivered vascular disrupting agents (VDAs): use of TNF-alpha conjugated gold nanoparticles for multimodal cancer therapy.

Surgery, radiation and chemotherapy remain the mainstay of current cancer therapy. However, treatment failure persists due to the inability to achieve complete local control of the tumor and curtail metastatic spread. Vascular disrupting agents (VDAs) are a class of promising systemic agents that are known to synergistically enhance radiation, chemotherapy or thermal treatments of solid tumors. Unfortunately, there is still an unmet need for VDAs with more favorable safety profiles and fewer side effects. Recent work has demonstrated that conjugating VDAs to other molecules (polyethylene glycol, CNGRCG peptide) or nanoparticles (liposomes, gold) can reduce toxicity of one prominent VDA (tumor necrosis factor alpha, TNF-α). In this report, we show the potential of a gold conjugated TNF-α nanoparticle (NP-TNF) to improve multimodal cancer therapies with VDAs. In a dorsal skin fold and hindlimb murine xenograft model of prostate cancer, we found that NP-TNF disrupts endothelial barrier function and induces a significant increase in vascular permeability within the first 1-2 h followed by a dramatic 80% drop in perfusion 2-6 h after systemic administration. We also demonstrate that the tumor response to the nanoparticle can be verified using dynamic contrast-enhanced magnetic resonance imaging (MRI), a technique in clinical use. Additionally, multimodal treatment with thermal therapies at the perfusion nadir in the sub- and supraphysiological temperature regimes increases tumor volumetric destruction by over 60% and leads to significant tumor growth delays compared to thermal therapy alone. Lastly, NP-TNF was found to enhance thermal therapy in the absence of neutrophil recruitment, suggesting that immune/inflammatory regulation is not central to its power as part of a multimodal approach. Our data demonstrate the potential of nanoparticle-conjugated VDAs to significantly improve cancer therapy by preconditioning tumor vasculature to a secondary insult in a targeted manner. We anticipate our work to direct investigations into more potent tumor vasculature specific combinations of VDAs and nanoparticles with the goal of transitioning optimal regimens into clinical trials.

Thermal Processing of Biological Tissue at High Temperatures: Impact of Protein Denaturation and Water Loss on the Thermal Properties of Human and Porcine Liver in the Range 25–80 °C

Biothermal engineering applications impose thermal excursions on tissues with an ensuing biological outcome (i.e., life or death) that is tied to the molecular state of water and protein in the system. The accuracy of heat transfer models used to predict these important processes in turn depends on the kinetics and energy absorption of molecular transitions for both water and protein and the underlying temperature dependence of the tissue thermal properties. Unfortunately, a lack of tissue thermal property data in the literature results in an overreliance on property estimates. This work addresses these thermal property limitations in liver, a platform tissue upon which hyperthermic engineering applications are routinely performed and a test bed that will allow extension to further tissue property measurement in the future. Specifically, we report on the thermal properties of cadaveric human and porcine liver in the suprazero range between 25 °C to 80 °C for thermal conductivity and 25 °C to 85 °C for apparent specific heat. Denaturation and water vaporization are shown to reduce thermal conductivity and apparent specific heat within the samples by up to 20% during heating. These changes in thermal properties significantly altered thermal histories during heating compared to conditions when properties were assumed to remain constant. These differences are expected to alter the biological outcome from heating as well.

RF heating of magnetic nanoparticles improves the thawing of cryopreserved biomaterials

While vitrified cryopreservation holds great promise, practical application has been limited to smaller systems (cells and thin tissues) due to diffusive heat and mass transfer limitations, which are typically manifested as devitrification and cracking failures during thaw. Here then we describe a new approach for rapidly and uniformly heating cryopreserved biospecimens with radiofrequency (RF) excited magnetic nanoparticles (mNPs). Importantly, heating rates can be increased several fold over conventional boundary heating techniques and are independent of sample size. Initial differential scanning calorimetry studies indicate that the addition of the mNPs has minimal impact on the freeze-thaw behavior of the cryoprotectant systems themselves. Then proof-of-principle experiments in aqueous and cryoprotectant solutions demonstrate the ability to heat at rates high enough to mitigate or eliminate devitrification (hundreds of °C/min) and scaled heat transfer modeling is used to illustrate the potential of this innovative approach. Finally, X-ray micro-computed-tomography (micro-CT) is investigated as a planning and quality control tool, where the density-based measurements are able to quantify changes in cryoprotectant concentration, mNP concentration, and the frozen state (i.e. crystallized versus vitrified).

Cooling rate dependent biophysical and viability response shift with attachment state in human dermal fibroblast cells.

While studies on the freezing of cells in suspension have been carried out extensively, corresponding studies with cells in the attached state and in tissue or tissue-equivalents are less developed. As attachment is a hallmark of the tissue state it is important to understand its impact on biophysics and viability to better apply freezing towards tissue preservation. The current study reports on observed biophysical response changes observed during freezing human dermal fibroblasts in suspension, attached cell, and fibrin tissue-equivalent models. Specifically, intracellular ice formation is shown to increase and dehydration is inferred to increase from suspension to attached systems. Biophysical model parameters fit to these experimental observations reflect the higher kinetics in the attached state. Post-thaw viability values from fast cooling rates were higher for suspension systems, and correlated well with the amount of IIF observed. On the other hand, viability values from slow cooling rates were higher for attached systems, although the degree of dehydration was predicted to be comparable to suspension cells. This disconnect between biophysics and viability predictions at slow rates clearly requires further investigation as it runs counter to our current understanding of dehydration injury in cells. This may suggest a possible protective effect of the attachment state on cell systems.

Calorimetric measurement of water transport and intracellular ice formation during freezing in cell suspensions

The current study presents a new and novel analysis of heat release signatures measured by a differential scanning calorimeter (DSC) associated with water transport (WT), intracellular ice formation (IIF) and extracellular ice formation (EIF). Correlative cryomicroscopy experiments were also performed to validate the DSC data. The DSC and cryomicroscopy experiments were performed on human dermal fibroblast cells (HDFs) at various cytocrit values (0-0.8) at various cooling rates (0.5-250 °C/min). A comparison of the cryomicroscopy experiments with the DSC analysis show reasonable agreement in the water transport (cellular dehydration) and IIF characteristics between both the techniques with the caveat that IIF measured by DSC lagged that measured by cryomicroscopy. This was ascribed to differences in the techniques (i.e. cell vs. bulk measurement) and the possibility that not all IIF is associated with visual darkening. High and low rates of 0.5 °C/min and 250 °C/min were chosen as HDFs did not exhibit significant IIF or WT at each of these extremes respectively. Analysis of post-thaw viability data suggested that 10 °C/min was the presumptive optimal cooling rate for HDFs and was independent of the cytocrit value. The ratio of measured heat values associated with IIF (q(IIF)) to the total heat released from both IIF and water transport or from the total cell water content in the sample (q(CW)) was also found to increase as the cooling rate was increased from 10 to 250 °C/min and was independent of the sample cytocrit value. Taken together, these observations suggest that the proposed analysis is capable of deconvolving water transport and IIF data from the measured DSC latent heat thermograms in cell suspensions during freezing.

A Micro-Thermal Sensor for Focal Therapy Applications

There is an urgent need for sensors deployed during focal therapies to inform treatment planning and in vivo monitoring in thin tissues. Specifically, the measurement of thermal properties, cooling surface contact, tissue thickness, blood flow and phase change with mm to sub mm accuracy are needed. As a proof of principle, we demonstrate that a micro-thermal sensor based on the supported “3ω” technique can achieve this in vitro under idealized conditions in 0.5 to 2 mm thick tissues relevant to cryoablation of the pulmonary vein (PV). To begin with “3ω” sensors were microfabricated onto flat glass as an idealization of a focal probe surface. The sensor was then used to make new measurements of ‘k’ (W/m.K) of porcine PV, esophagus, and phrenic nerve, all needed for PV cryoabalation treatment planning. Further, by modifying the sensor use from traditional to dynamic mode new measurements related to tissue vs. fluid (i.e. water) contact, fluid flow conditions, tissue thickness, and phase change were made. In summary, the in vitro idealized system data presented is promising and warrants future work to integrate and test supported “3ω” sensors on in vivo deployed focal therapy probe surfaces (i.e. balloons or catheters).

In vivo detection of the effects of preconditioning on LNCaP tumors by a TNF-α nanoparticle construct using MRI.

The outcome of systemic and local therapies (e.g. chemotherapy, radiotherapy, surgery, focal ablation) for prostate cancer can be significantly improved by using tumor‐specific adjuvants prior to treatment (“preconditioning”). We propose to use dynamic contrast enhanced magnetic resonance imaging (DCE‐MRI) to monitor the in vivo response of a mouse model of prostate cancer treated with a vascular disruptive agent, TNF‐α, delivered on a gold nanoparticle (NP‐TNF). Six male nude mice bearing 4–5 week old LNCaP tumors were scanned at 9.4 T. DCE‐MRI was performed two days before and 4–5 h after treatment with NP‐TNF. An intraperitoneal (i.p.) bolus of gadolinium‐DTPA (Gd) was administered and contrast enhancement was measured for 90 min. Concentration–time curves of Gd were calculated and the area under the Gd curve (AUGC) was determined pre‐ and post‐treatment. NP‐TNF treatment caused an increase in contrast uptake in tumors. Interestingly, the early concentration (10 min post Gd bolus i.p.) was similar in both untreated and treated conditions; however, 90 min after injection, [Gd] was 3.4 times higher than before treatment. AUGC doubled from (11 ± 6) [Gd] × min before treatment to (22 ± 9) [Gd] × min after treatment. An increase in signal enhancement was also observed in the muscle but to a lesser degree. We also evaluated the kinetics of intravenous Gd administration in mice bearing a jugular vein catheter to mimic the delivery method used in clinical trials. The overall treatment effects were independent of the delivery pathway of the contrast agent. In conclusion, we show that DCE‐MRI is suitable to detect changes associated with a vascular disruptive agent in a mouse model of prostate cancer. The ability to characterize the effects of nanoparticle therapy in vivo with non‐destructive methods is important, as such compounds, in combination with treatment strategies, are progressing towards clinical trials. Copyright

Spectroscopic and Calorimetric Evaluation of Chemically Induced Protein Denaturation in HuH-7 Liver Cancer Cells and Impact on Cell Survival

Solid tumors such as hepatocellular carcinoma are very often not amenable to chemotherapy and radiotherapy. Local ablation methods, including chemical ablation with absolute ethanol, are therefore an option for treatment but lack of information about the mechanism of devitalization leading to cell death is a hindrance to further adoption. Systemic toxicity also has limited the amount of ethanol that can be used in a single treatment session. Therefore we evaluated the mechanism of urea, a denaturant with little or no systemic toxicity, for potential use in chemical ablation. In this study we report on the use of three methods to analyze the effects in cell culture with a view towards eventual clinical application. Human hepatoma HuH-7 cells were analyzed at several time points after treatment using FTIR, DSC, and Raman microspectroscopy based on MTT and PI-exclusion viability assays. Time course fractional denaturation data plotted against viability show that a 50% viability drop occurs after only a 10–20% drop in overall protein denaturation. Other methods of cell death such as apoptosis may also be operative, but this result implies that protein denaturation is one of the major mechanisms of cell death. This is in line with what has been previously suggested for purely thermal methods, and opens the way to mechanism-based improvements in chemical ablation of solid tumors.