A Multimodality Image Guided Precision Radiation Research Platform: Integrating X-ray, Bioluminescence, and Fluorescence Tomography With Radiation Therapy.

PURPOSE: Small animal irradiation is crucial to the investigation of radiobiological mechanisms. The paradigm of clinical radiation therapy is trending toward high-precision, stereotactic treatment. However, translating this scheme to small animal irradiation is challenging owing to the lack of high-quality image guidance. To overcome this obstacle, we developed a multimodality image guided precision radiation platform. METHODS AND MATERIALS: The platform consists of 4 modules: x-ray computed tomography (CT), bioluminescence tomography (BLT), fluorescence molecular tomography (FMT), and radiation therapy. CT provides animal anatomy and material density for radiation dose calculation, as well as body contour for BLT and FMT reconstruction. BLT and FMT provide tumor localization to guide radiation beams and molecular activity to evaluate treatment outcome. Furthermore, we developed a Monte Carlo-based treatment planning system (TPS) for 3-dimensional dose calculation, calibrated it using radiochromic films sandwiched in a water-equivalent phantom, and validated it using in vivo dosimeters surgically implanted into euthanized mice (n = 4). Finally, we performed image guided irradiation on mice bearing orthotopic breast and prostate tumors and confirmed radiation delivery using γH2AX histology. RESULTS: The Monte Carlo-based TPS was successfully calibrated by benchmarking simulation dose against film measurement. For in vivo dosimetry measured in the euthanized mice, the average difference between the TPS calculated dose and measured dose was 3.86% ± 1.12%. Following the TPS-generated treatment plan, we successfully delivered 20 Gy dose to an animal bearing an orthotopic prostate tumor using 4 BLT-guided radiation beams and 5 Gy dose to an animal bearing an orthotopic breast tumor using a single FMT-guided radiation beam. γH2AX histology presented significantly more DNA damage in irradiated tumors and thus validated the dose delivery accuracy. CONCLUSIONS: Combined with Monte Carlo TPS, this multimodality CT/BLT/FMT image guided small animal radiation platform can specifically localize tumors, accurately calculate dose distribution, precisely guide radiation delivery, and molecularly evaluate treatment response. It provides an advanced toolset for radiobiology and translational cancer research.

Data for optimizing Gamma Knife radiosurgery using the shot within shot technique.

The tables included in this article will allow the user to implement shot within shot optimization for Gamma Knife radiosurgery planning and delivery. The method is intended to reduce treatment time when treating small to medium sized brain metastasis. The tables were previously developed by extracting profiles from Gamma Plan for three collimator settings and modeling their behavior when combined or prescribed at different isodose lines. For a given target size, the tables represent the optimal selection of shot weighting and prescription isodose line to reduce beam on time while maintaining an acceptable dose gradient. The method was recently validated in a large patient cohort and the data is this study is related to the research article titled "Clinical evaluation of shot within shot optimization for Gamma Knife radiosurgery planning and delivery" (Johnson et al., in press).

Clinical Evaluation of Shot-Within-Shot Optimization for Gamma Knife Radiosurgery Planning and Delivery.

OBJECTIVE: Shot-within-shot (SWS) optimization is a new planning technique that relies on various combinations of shot weighting and prescription isodose line (IDL) to reduce beam-on time. The method differs from other planning techniques that incorporate mixed collimation, multiple stereotactic coordinates, and traditionally low prescription IDLs (<60%). In this work, we evaluate the percentage of brain metastasis for which the method can be applied, the magnitude of the resultant time savings, and the possible tradeoffs in plan quality. METHODS: A retrospective analysis was performed on 75 patients treated for 241 metastatic lesions in the brain. For each lesion, the original planning metrics related to target coverage, conformity, gradient, and beam-on time were recorded. A subset of lesions were selected for replanning using the SWS technique based on size, shape, and proximity to critical structures. Two replans were done, a reference plan was prescribed at the 50% IDL, and an optimized plan was prescribed at an IDL typically >50%. Planning metrics were then compared among the original plan and the 2 replans. RESULTS: More than a third (39%) of the brain metastases were eligible for the SWS technique. For these lesions, the differences between the original plan and reference SWS plan were as follows: ΔV12Gy < 0.5 cc in 93% of cases, ΔV12Gy < 0.5 cc in 100% of cases, Δselectivity < 0.1 in 79% of cases. Negligible differences were seen between the 2 replans in terms of Δselectivity and ΔV12Gy; ΔGI < 5% in 99% of cases. After optimization, beam-on time was reduced by 25%-30% in approximately 40%-50% of eligible lesions when compared with the reference SWS plan (ΔTmax = 42%). In comparison with the original plan, beam-on time was reduced even further, ΔT > 50% in 20% of cases (ΔTmax = 70%). CONCLUSIONS: This work demonstrates clinically that optimization using the shot-within-shot technique can reduce beam-on time without degrading treatment plan quality.

Technical Note: Minimizing geometrical uncertainties of cylindrical well-type ionization chamber measurements: There is an optimal chamber length.

PURPOSE: The response of well-type ionization chambers used, for example, in brachytherapy and nuclear medicine, depends on the location of the source. In cases where the source length is variable (typically in nuclear medicine), it is also dependent on length of the source. Here, the combined effect on chamber sensitivity of both source position and length is investigated in detail. This analysis is important if nominal values for source location and length are prescribed as (arbitrary but fixed) values in order to precisely define a chamber's sensitivity. During measurement, the actual values for source location and length can deviate from the nominal ones, altering sensitivity and thus giving rise to measurement uncertainties which, in turn, directly affect the doses administered to patients. Our aim is to investigate these uncertainties and minimize them with an optimized ion chamber design. METHODS: An analytical model for the chamber response is used to describe the variation of chamber sensitivity with respect to the two parameters, source position and length. The influence of the relative magnitude of uncertainty in both parameters is also accounted for. The effect of their combined variation on chamber sensitivity is required to be minimal and, employing differential geometry tools, a relationship is derived between them and the optimal height of the ionization chamber's sensitive volume. RESULTS: This relationship provides the chamber height h which minimizes its response variation for given nominal value of source location (quantified as insertion depth d) and prescribed source length l: h=2d-ρ(ρ)l, where ρ = Î´d/δl is defined as the quotient of uncertainty in insertion depth, δd, and uncertainty in source length, δl, and ρρ=4/9+ρ21+4ρ2, so that the optimal ionization chamber length varies between h=2d-12l and h=2d-23l. Alternatively, if h is given, suitable combinations of d and l can be deduced. CONCLUSIONS: The analysis presented here provides a tool for reducing the uncertainty budget of any cylindrically designed ionization chambers utilized for measuring extended on-axis sources. In particular, these results can be applied to a calibration-type ionization chamber design recently proposed for the cross-calibration of unsealed radionuclides.

The Phase Behavior and Organization of Sphingomyelin/Cholesterol Membranes: A Deuterium NMR Study.

We have studied the dependence of the phase and domain characteristics of sphingomyelin (SM)/cholesterol model membranes on sterol content and temperature using deuterium nuclear magnetic resonance. NMR spectra of N-palmitoyl(D31)-D-erythro-sphingosylphosphorylcholine (PSM-d31) were taken for temperatures from 25 to 70°C and cholesterol concentrations of 0-40%. Analogous experiments were performed using 1-palmitoyl,2-palmitoyl(D31)-sn-glycero-3-phosphocholine (DPPC-d31)/cholesterol membranes to carefully compare the data obtained using palmitoyl chains that have similar "kinked" conformations. The constructed phase diagrams exhibit both solid-ordered (so) + liquid-ordered (lo) and liquid-disordered (ld) + lo phase-coexistence regions with a clear three-phase line. Macroscopic (micron-sized) coexistence of ld and lo phases was not observed; instead, line-broadening in the ld+lo region was characterized by intermediate exchange of lipids between the two types of domains. The length scales associated with the domains were estimated to be 75-150 nm for PSM-d31/cholesterol and DPPC-d31/cholesterol model membranes.

Constrained Versus Free Cholesterol in DPPC Membranes: A Comparison of Chain Ordering Ability Using Deuterium NMR.

We report here the first exploration of the nature of the hydrophobic region of bilayer membranes formed from sterol-modified phospholipids [Huang, Z.; Szoka, F. C., Sterol-Modified Phospholipids: Cholesterol and Phospholipid Chimeras with Improved Biomembrane Properties. J. Am. Chem. Soc. 2008, 130 (46), 15702-15712] & [Ding, J.; Starling, A. P.; East, J. M.; Lee, A. G., Binding Sites for Cholesterol on Ca(2+)-ATPase Studied by Using a Cholesterol-Containing Phospholipid. Biochemistry 1994, 33 (16), 4974-4979]. Using 2H NMR spectroscopy, we present our results for the phase behavior and acyl chain ordering of multilamellar vesicles (MLVs) of a sterol-modified phospholipid, 1-cholesterylhemisuccinoyl-2-palmitoyl(d31)-sn-glycero-3-phosphocholine (hereafter referred to as CholPPC-d31). We compared our results with the conformational order induced by cholesterol at various concentrations in 1-palmitoyl,2-palmitoyl(d31)-sn-glycero-3-phosphocholine (DPPC-d31)/cholesterol membranes. On the basis of the existing literature [Foglia, F.; Barlow, D. J.; Szoka, F. C.; Huang, Z.; Rogers, S. E.; Lawrence, M. J., Structural Studies of the Monolayers and Bilayers Formed by a Novel Cholesterol-Phospholipid Chimera. Langmuir 2011, 27 (13), 8275-8281], we expected to find that the deuterated palmitoyl chain in CholPPC-d31 membranes had an order parameter profile similar to the deuterated palmitoyl chain of sn-2 labeled DPPC-d31 in MLVs of a mixture of DPPC-d31 with 40 mol % unconstrained cholesterol. Our data indicate that the ordering ability of cholesterol in CholPPC is significantly reduced compared to free cholesterol in DPPC. This result emphasizes that cholesterol molecules must be free to move in the bilayers to reach their maximum ordering ability. In other words, when compared to unconstrained cholesterol, the constrained cholesterol moiety in CholPPC causes nonoptimal chain packing.

Insights into sphingolipid miscibility: separate observation of sphingomyelin and ceramide N-acyl chain melting.

Ceramide produced from sphingomyelin in the plasma membrane is purported to affect signaling through changes in the membrane's physical properties. Thermal behavior of N-palmitoyl sphingomyelin (PSM) and N-palmitoyl ceramide (PCer) mixtures in excess water has been monitored by ²H NMR spectroscopy and compared to differential scanning calorimetry (DSC) data. The alternate use of either perdeuterated or proton-based N-acyl chain PSM and PCer in our ²H NMR studies has allowed the separate observation of gel-fluid transitions in each lipid in the presence of the other one, and this in turn has provided direct information on the lipids' miscibility over a wide temperature range. The results provide further evidence of the stabilization of the PSM gel state by PCer. Moreover, overlapping NMR and DSC data reveal that the DSC-signals parallel the melting of the major component (PSM) except at intermediate (20 and 30 mol %) fractions of PCer. In such cases, the DSC endotherm reports on the presumably highly cooperative melting of PCer. Up to at least 50 mol % PCer, PSM and PCer mix ideally in the liquid crystalline phase; in the gel phase, PCer becomes incorporated into PSM:PCer membranes with no evidence of pure solid PCer.

Influence of the nature of the sterol on the behavior of palmitic acid/sterol mixtures and their derived liposomes.

The phase behavior of mixtures formed with palmitic acid (PA) and one of the following sterols (dihydrocholesterol, ergosterol, 7-dehydrocholesterol, stigmasterol and stigmastanol), in a PA/sterol molar ratio of 3/7, has been characterized by IR and (2)H NMR spectroscopy at different pH. Our study shows that it is possible to form liquid-ordered (lo) lamellar phases with these binary non-phospholipid mixtures. The characterization of alkyl chain dynamics of PA in these systems revealed the large ordering effect of the sterols. It was possible to extrude these systems, using standard extrusion techniques, to form large unilamellar vesicles (LUVs), except in the case of ergosterol-containing mixture. The resulting LUVs displayed a very limited passive permeability consistent with the high sterol concentration. In addition, the stability of these PA/sterol self-assembled bilayers was also found to be pH-sensitive, therefore, potentially useful as nanovectors. By examining different sterols, we could establish some correlations between the structure of these bilayers and their permeability properties. The structure of the side chain at C17 of the sterol appears to play a prime role in the mixing properties with fatty acid.

Interaction of a charged polymer with zwitterionic lipid vesicles.

The interaction between polyethylenimine (PEI) and phospholipid bilayers plays an important role in several biophysical applications such as DNA transfection of target cells. Despite considerable investigation into the nature of the interaction between PEI and phospholipid bilayers, the physical process remains poorly understood. In this paper, we study the impact of PEI on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles as a function of salt concentration using several techniques including dynamic (DLS) and static (SLS) light scattering, differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR). At low salt concentration, vesicles aggregate, leading to the formation of stable clusters whose final size depends on the PEI concentration. At high salt concentration the system does not aggregate; DSC and NMR data reveal that the PEI penetrates into the bilayer, and SLS measurements are consistent with PEI crossing the bilayer. The transfectional ability of PEI is discussed in terms of these results.