Active targeting of block copolymer micelles with trastuzumab Fab fragments and nuclear localization signal leads to increased tumor uptake and nuclear localization in HER2-overexpressing xenografts.

Block copolymer micelles (BCMs) have been employed as effective drug delivery systems to solid tumors by virtue of their capacity to transport large therapeutic payloads and passively target tumor sites. Active targeting of nanoparticles (NPs) has been exploited as a means to increase the therapeutic efficacy of NP-based drugs by promoting their delivery to cellular sites of action. Effective whole tumor accumulation and cellular uptake constitute key objectives in the success of preclinical drug formulations, although they have seldom been investigated concurrently in vivo. The current study aims to elucidate the in vivo fate of 31-nm-sized block copolymer micelles (BCMs) targeted to the nucleus of HER2-overexpressing breast cancer cells. Pharmacokinetics, biodistribution, tumor uptake, and intratumoral distribution of BCMs were investigated in mice bearing subcutaneous BT-474 and MDA-MB-231 xenografts expressing high and low levels of HER2, respectively. Radiolabeling with (111)indium enabled quantitative assessment of BCM distribution at the whole body, tissue, and cellular levels. Surface-grafted trastuzumab Fab fragments (TmAb-Fab) facilitated binding and internalization of BCMs by HER2-positive breast cancer cells, while synthetic 13-mer nuclear localization signal (NLS) peptides conjugated to the TmAb-Fab conferred nuclear translocation capability. Active targeting of BCMs led to a 5-fold increase in tumor uptake in HER2-overexpressing BT-474 tumors, alongside a correspondingly greater level of cellular uptake and nuclear localization, relative to the nontargeted formulations. This study distinctively highlights the quantitative evaluation of active targeting on tumor, cellular and subcellular uptake of BCMs and presents a promising platform for the effective delivery of chemo- and/or radiotherapy in vivo.

Hyperthermia-induced doxorubicin delivery from thermosensitive liposomes via MR-HIFU in a pig model.

PURPOSE: Encapsulation of cytotoxic drugs for a localized release is an effective way to increase the therapeutic window of such agents. In this article we present the localized release of doxorubicin (DOX) from phosphatidyldiglycerol (DPPG2) based thermosensitive liposomes using MR-HIFU mediated hyperthermia in a swine model. MATERIALS AND METHODS: German landrace pigs of weights between 37.5 and 53.5 kg received a 30-min infusion of DOX containing thermosensitive liposomes (50 mg DOX/m2). The pigs' biceps femoris was treated locally in two separate target areas with mild hyperthermia using magnetic resonance guided high intensity focused ultrasound, starting 10 min and 60 min after initiation of the infusion, respectively. The pharmacokinetics and biodistribution of DOX were determined and an analysis of the treatment parameters' influence was performed. RESULTS: Compared to untreated tissue, we found a 15-fold and a 7-fold increase in DOX concentration in the muscle volumes that had undergone hyperthermia starting 10 min and 60 min after the beginning of the infusion, respectively. The pharmacokinetic analysis showed a prolonged circulation time of DOX and a correlation between the AUC of extra-liposomal DOX in the bloodstream and the amount of DOX accumulated in the target tissue. CONCLUSIONS: We have demonstrated a workflow for MR-HIFU hyperthermia drug delivery that can be adapted to a clinical setting, showing that HIFU-hyperthermia is a suitable method for local drug release of DOX using DPPG2 based thermosensitive liposomes in stationary targets. Using the developed pharmacokinetic model, an optimization of the drug quantity deposited in the target via the timing of infusion and hyperthermia should be possible.

Value of spectral detector computed tomography for assessment of pancreatic lesions.

PURPOSE: Dual energy CT (DECT) can contribute to the diagnosis of benign and malignant pancreatic lesions. This study examined whether a novel, detector-based spectral CT scanner (SDCT) may improve subjective assessment of different types of pancreatic lesions and if various quantitative maps may improve lesion contrast and differentiation. MATERIALS AND METHODS: 61 consecutive patients who underwent clinical, contrast-agent enhanced, abdominal SDCT scans and showed pancreatic lesions of different origins were included. Subjective image analysis was performed by two readers who assessed image quality, lesion conspicuity and diagnostic confidence on 5-point Likert scales for conventional polyenergetic reconstructions (polyE), virtual monoenergetic images (monoE), virtual non-contrast images, iodine density, iodine overlay, and Z effective (Zeff) maps. Two readers acquired quantitative values from these maps ROI-based from which contrast-to-noise and lesion-to-parenchyma ratios were calculated. RESULTS: MonoE images at low keV levels yielded highest Likert scores regarding lesion conspicuity and reader confidence; iodine overlays facilitated lesion delineation. Inter-observer agreement ranged between substantial and excellent (kappa values 0.73-0.81). Contrast-to-noise-ratios for low keV monoE images were significantly higher, compared to polyE images (e.g. monoE 40 keV p < 0.0001). Marked overlap between PDAC and miscellaneous non-PDAC lesions was present in various spectral reconstructions. CONCLUSIONS: In line with previous studies, monoE images at low keV levels and iodine overlay maps facilitated subjective lesion delineation which was substantiated by the quantitative analysis. Hence, spectral detector CT improves pancreatic lesion conspicuity, while its value for lesion differentiation needs to be further evaluated in larger study cohorts.

4D flow MRI for the analysis of celiac trunk and mesenteric artery stenoses.

PURPOSE: This study aims to assess the feasibility of 4D flow MRI measurements in complex vascular territories; namely, the celiac artery (CA) and superior mesenteric artery (SMA). MATERIALS AND METHODS: In this prospective study, 22 healthy volunteers and 10 patients were scanned at 3 T. Blood flow parameters were compared between healthy volunteers and patients with stenosis of the CA and/or SMA as a function of stenosis grade characterized by prior contrast-enhanced computed tomography (CE-CT). The 4D flow MRI acquisition covered the CA, SMA and adjusting parts of the abdominal aorta (AO). Measurements of velocity- (peak velocity [PV], average velocity [AV]) and volume-related parameters (peak flow [PF], stroke volume [SV]) were conducted. Further, stenosis grade and wall shear stress in the CA, SMA and AO were evaluated. RESULTS: In patients, prior evaluation by CE-CT revealed 11 low- and 5 mid-grade stenoses of the CA and/or SMA. PV and AV were significantly higher in patients than in healthy volunteers [PV: p < 0.0001; AV: p = 0.03, p < 0.001]. PF and SV did not differ significantly between healthy volunteers and patients; however, a trend towards lower PF and SV could be detected in patients with mid-grade stenoses. Comparison of 4D flow MRI with CE-CT revealed a strong positive correlation in estimated degree of stenosis (CA: r = 0.86, SMA: r = 0.98). Patients with mid-grade stenoses had a significantly higher average WSS magnitude (AWM) than healthy volunteers (p = 0.02). CONCLUSION: This feasibility study suggests that 4D flow MRI is a viable technique for the evaluation of complex flow characteristics in small vessels such as the CA and SMA. 4D flow MRI approves comparable to the morphologic assessment of complex vascular territories using CE-CT but, in addition, offers the functional evaluation of flow parameters that goes beyond the morphology.

The challenges facing block copolymer micelles for cancer therapy: In vivo barriers and clinical translation.

The application of block copolymer micelles (BCMs) in oncology has benefitted from advances in polymer chemistry, drug formulation and delivery as well as in vitro and in vivo biological models. While great strides have been made in each of these individual areas, there remains some disappointment overall, citing, in particular, the absence of more BCM formulations in clinical evaluation and practice. In this review, we aim to provide an overview of the challenges presented by in vivo systems to the effective design and development of BCMs. In particular, the barriers posed by systemic administration and tumor properties are examined. The impact of critical features, such as the size, stability and functionalization of BCMs is discussed, while key pre-clinical endpoints and models are critiqued. Given clinical considerations, we present this work as a means to stimulate a renewed focus on the unique chemical versatility bestowed by BCMs and a measured grasp of representative in vitro and in vivo models.

Development of a liposome formulation for improved biodistribution and tumor accumulation of pentamidine for oncology applications.

Pentamidine isethionate, widely used for the treatment of parasitic infections, has shown strong anticancer activity in cancer cells and models of melanoma and lung cancer. Systemic administration of pentamidine is associated with serious toxicities, particularly renal, affecting as many as 95% of patients (O'Brien et al., 1997). This work presents the development of a liposome pentamidine formulation for greater tumor accumulation and lower drug exposure to vulnerable tissues. Liposomes formulated with saturated/unsaturated phospholipids of different chain lengths, varying cholesterol content, and surface PEG were explored to understand the effects of such variations on drug release, encapsulation efficiency, stability and in vivo performance. Saturated phospholipids with longer chain lengths, higher cholesterol content and PEG resulted in greater stability. The optimal formulation obtained showed significantly lower clearance rate (3.6 ± 1.2 mL/h/Kg) and higher AUC0-inf (348 ± 31 µmol/L × h) in vivo when compared to free drug (414 ± 138 mL/h/Kg and 2.58 ± 0.74 µmol/L × h, respectively). In tumor-bearing mice, liposomal delivery decreased kidney drug levels by up to 5-fold at 6 and 24h post-administration. Tumor drug exposure was up to 12.7-fold greater with liposomal administration compared to free drug. Overall, the liposomal pentamidine formulation developed has significant potential for the treatment of solid tumors.

Spatial and temporal mapping of heterogeneity in liposome uptake and microvascular distribution in an orthotopic tumor xenograft model.

Existing paradigms in nano-based drug delivery are currently being challenged. Assessment of bulk tumor accumulation has been routinely considered an indicative measure of nanomedicine potency. However, it is now recognized that the intratumoral distribution of nanomedicines also impacts their therapeutic effect. At this time, our understanding of the relationship between the bulk (i.e., macro-) tumor accumulation of nanocarriers and their intratumoral (i.e., micro-) distribution remains limited. Liposome-based drug formulations, in particular, suffer from diminished efficacy in vivo as a result of transport-limiting properties, combined with the heterogeneous nature of the tumor microenvironment. In this report, we perform a quantitative image-based assessment of macro- and microdistribution of liposomes. Multi-scalar assessment of liposome distribution was enabled by a stable formulation which co-encapsulates an iodinated contrast agent and a near-infrared fluorescence probe, for computed tomography (CT) and optical microscopy, respectively. Spatio-temporal quantification of tumor uptake in orthotopic xenografts was performed using CT at the bulk tissue level, and within defined sub-volumes of the tumor (i.e., rim, periphery and core). Tumor penetration and relative distribution of liposomes were assessed by fluorescence microscopy of whole tumor sections. Microdistribution analysis of whole tumor images exposed a heterogeneous distribution of both liposomes and tumor vasculature. Highest levels of liposome uptake were achieved and maintained in the well-vascularized tumor rim over the study period, corresponding to a positive correlation between liposome and microvascular density. Tumor penetration of liposomes was found to be time-dependent in all regions of the tumor however independent of location in the tumor. Importantly, a multi-scalar comparison of liposome distribution reveals that macro-accumulation in tissues (e.g., blood, whole tumor) may not reflect micro-accumulation levels present within specific regions of the tumor as a function of time.

Image-based analysis of the size- and time-dependent penetration of polymeric micelles in multicellular tumor spheroids and tumor xenografts.

While the heightened tumor accumulation of systemically administered nanomedicines relative to conventional chemotherapeutic agents has been well established, corresponding improvements in therapeutic efficacy have often been incommensurate. This observation may be attributed to the limited exposure of cancer cells to therapy due to the heterogeneous intratumoral distribution and poor interstitial penetration of nanoparticle-based drug delivery systems. In the present work, the spatio-temporal distribution of block copolymer micelles (BCMs) of different sizes was evaluated in multicellular tumor spheroids (MCTS) and tumor xenografts originating from human cervical (HeLa) and colon (HT29) cancer cells using image-based, computational techniques. Micelle penetration was found to depend on nanoparticle size, time as well as tumor and spheroid cell line. Moreover, spheroids demonstrated the capacity to predict relative trends in nanoparticle interstitial transport in tumor xenografts. Overall, techniques are presented for the assessment of nanoparticle distribution in spheroids and xenografts and used to evaluate the influence of micelle size and cell-line specific tissue properties on micelle interstitial penetration.