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Background: Medical images play an important role in diagnosis and treatment of pediatric solid tumors. The field of radiology, pathology, and other image-based diagnostics are getting increasingly important and advanced. This indicates a need for advanced image processing technology such as Deep Learning (DL)., Aim: Our review focused on the use of DL in multidisciplinary imaging in pediatric surgical oncology., Methods: A search was conducted within three databases (Pubmed, Embase, and Scopus), and 2056 articles were identified. Three separate screenings were performed for each identified subfield., Results: In total, we identified 36 articles, divided between radiology (n = 22), pathology (n = 9), and other image-based diagnostics (n = 5). Four types of tasks were identified in our review: classification, prediction, segmentation, and synthesis. General statements about the studies'' performance could not be made due to the inhomogeneity of the included studies. To implement DL in pediatric clinical practice, both technical validation and clinical validation are of uttermost importance., Conclusion: In conclusion, our review provided an overview of all DL research in the field of pediatric surgical oncology. The more advanced status of DL in adults should be used as guide to move the field of DL in pediatric oncology further, to keep improving the outcomes of children with cancer., (© 2025 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)

Background/Objectives : Integrating the cytotoxic drug busulfan into a high-dose chemotherapy regimen prior to autologous hematopoietic stem cell rescue in patients with high-risk neuroblastoma has improved the survival of children battling this deadly disease. Busulfan-induced toxicities can, however, be severe. Here, we describe the diagnosis and successful treatment of acute pulmonary injury by total-body-weight-adjusted busulfan therapy in two children with high-risk neuroblastoma. Case series : Patient 1 developed life-threatening biphasic acute respiratory failure on days +60 and +100 after busulfan therapy, requiring intubation and invasive mechanical ventilation. Despite intensive anti-inflammatory and immunomodulatory therapy, including systemic corticosteroids, topical inhalation regimens, azithromycin, nintedanib and extracorporal photopheresis, patient 1 required extended intensive care measures and non-invasive respiratory support for a total of 20 months. High-resolution computed tomography showed diffuse intra-alveolar and interstitial patterns. Patient 2 developed partial respiratory failure with insufficient oxygen saturation and dyspnea on day +52 after busulfan therapy. Symptoms were resolved after 6 months of systemic corticosteroids, topical inhalation regimens and azithromycin. High-resolution computed tomography showed atypical pneumonic changes with ground-glass opacities. While both patients fully recovered without evidence of pulmonary fibrosis, cancer therapy had to be paused and then modified until full recovery from busulfan-induced lung injury. Conclusions : Busulfan-induced lung injury requires prompt diagnosis and intervention. Symptoms and signs are nonspecific and difficult to differentiate from other causes. Therapeutic busulfan drug level monitoring and the identification of patients at risk for drug overdosing through promoter polymorphisms in the glutathione S-transferase alpha 1 gene encoding the main enzyme in busulfan metabolism are expected to reduce the risk of busulfan-induced toxicities.

Purpose: To compare suprapubic access (SPA) and transurethral catheterization (TUC) in voiding cystourethrogram (VCUG)., Methods: Retrospective single-center evaluation of 311 VCUG performed in male patients under 12 years of age. Two study groups were built based on the bladder access method. TUC was performed in 213 patients, whereas 98 received SPA. The groups were compared regarding the procedural switch rate, the complication rate, radiation parameters, the amount of contrast media applied and the examination quality. Complications were graded in minor (contrast leakage, premature termination of the examination) and major (fever, urinary tract infection, bladder perforation). Fluoroscopy time and radiation parameters were compared. Examination quality was assessed based on the satisfactory acquisition of fluoroscopic images using a four-point Likert scale., Results: In 9% of the SPA examinations a method switch to TUC was necessary. The minor complication rate was 1.9% for TUC and 35.7% for SPA ( p < 0.001). The major complication rate was 0.9% for TUC and 2% for SPA ( p > 0.05). Mean fluoroscopy time and radiation dose were significantly lower in TUC (TUC, 26 ± 19 s, 0.6 ± 1.2 µGy·m 2 ; SPA, 38 ± 33 s, 1.7 ± 2.9 µGy·m 2 ; p = 0.01/0.001). There was no significant difference regarding the amount of contrast media applied (TUC, 62 ± 40 mL; SPA, 66 ± 41 mL; p > 0.05) and the examination quality with full diagnostic quality achieved in 88% of TUC and 89% of SPA examinations ( p > 0.05)., Conclusions: As TUC provides significantly lower radiation exposure and less periprocedural complications, it should be the primary bladder access route for VCUG in pediatric male patients.

Background The triple combination of the cystic fibrosis transmembrane regulator (CFTR) modulators elexacaftor, tezacaftor, and ivacaftor (hereafter, elexacaftor/tezacaftor/ivacaftor) has a positive effect on lung function in patients with cystic fibrosis (CF). Purpose To compare three-dimensional (3D) ultrashort echo time (UTE) MRI functional lung data to common functional lung parameters in assessing lung function in patients with CF undergoing elexacaftor/tezacaftor/ivacaftor therapy. Materials and Methods In this prospective feasibility study, 16 participants with CF consented to undergo pulmonary MRI with a breath-hold 3D UTE sequence at baseline (April 2018-June 2019) and follow-up (April-July 2021). Eight participants received elexacaftor/tezacaftor/ivacaftor after baseline, and eight participants with unchanged treatment served as the control group. Lung function was assessed with body plethysmography and lung clearance index (LCI). Image-based functional lung parameters, such as ventilation inhomogeneity and ventilation defect percentage (VDP), were calculated from signal intensity change between MRI scans at inspiration and expiration. Metrics at baseline and follow-up were compared within groups (permutation test), correlation was tested (Spearman rank correlation), and 95% CIs were calculated (bootstrapping technique). Results MRI ventilation inhomogeneity correlated with LCI at baseline ( r = 0.92, P < .001) and follow-up ( r = 0.81, P = .002). Mean MRI ventilation inhomogeneity (baseline, 0.74 ± 0.15 [SD]; follow-up, 0.64 ± 0.11; P = .02) and mean VDP (baseline, 14.1% ± 7.4; follow-up, 8.5% ± 3.3; P = .02) decreased from baseline to follow-up in the treatment group. Lung function was stable over time (mean LCI: 9.3 turnovers ± 4.1 at baseline vs 11.5 turnovers ± 7.4 at follow-up; P = .34) in the control group. In all participants, correlation of forced expiratory volume in 1 second with MRI ventilation inhomogeneity was good at baseline ( r = -0.61, P = .01) but poor during follow-up ( r = -0.06, P = .82). Conclusion Noncontrast 3D UTE lung MRI functional parameters of ventilation inhomogeneity and VDP can be used to assess lung function over time in patients with CF and can add regional information to established global parameters, such as LCI. © RSNA, 2023 Supplemental material is available for this article. See also the editorial by Iwasawa in this issue.

Background: Morphology and glenoid involvement determine the necessity of surgical management in scapula fractures. While being present in only a small share of patients with shoulder trauma, numerous classification systems have been in use over the years for categorization of scapula fractures. The purpose of this study was to evaluate the established AO/OTA classification in comparison to the classification system of Euler and Rüedi (ER) with regard to interobserver reliability and confidence in clinical practice., Methods: Based on CT imaging, 149 patients with scapula fractures were retrospectively categorized by two trauma surgeons and two radiologists using the classification systems of ER and AO/OTA. To measure the interrater reliability, Fleiss kappa (κ) was calculated independently for both fracture classifications. Rater confidence was stated subjectively on a five-point scale and compared with Wilcoxon signed rank tests. Additionally, we computed the intraclass correlation coefficient (ICC) based on absolute agreement in a two-way random effects model to assess the diagnostic confidence agreement between observers., Results: In scapula fractures involving the glenoid fossa, interrater reliability was substantial (κ = 0.722; 95% confidence interval [CI] 0.676-0.769) for the AO/OTA classification in contrast to moderate agreement (κ = 0.579; 95% CI 0.525-0.634) for the ER classification system. Diagnostic confidence for intra-articular fracture patterns was superior using the AO/OTA classification compared to ER (p < 0.001) with higher confidence agreement (ICC: 0.882 versus 0.831). For extra-articular fractures, ER (κ = 0.817; 95% CI 0.771-0.863) provided better interrater reliability compared to AO/OTA (κ = 0.734; 95% CI 0.692-0.776) with higher diagnostic confidence (p < 0.001) and superior agreement between confidence ratings (ICC: 0.881 versus 0.912)., Conclusions: The AO/OTA classification is most suitable to categorize intra-articular scapula fractures with glenoid involvement, whereas the classification system of Euler and Rüedi appears to be superior in extra-articular injury patterns with fractures involving only the scapula body, spine, acromion and coracoid process., (© 2022. The Author(s).)

Background: Functional lung MRI techniques are usually associated with time-consuming post-processing, where manual lung segmentation represents the most cumbersome part. The aim of this study was to investigate whether deep learning-based segmentation of lung images which were scanned by a fast UTE sequence exploiting the stack-of-spirals trajectory can provide sufficiently good accuracy for the calculation of functional parameters., Methods: In this study, lung images were acquired in 20 patients suffering from cystic fibrosis (CF) and 33 healthy volunteers, by a fast UTE sequence with a stack-of-spirals trajectory and a minimum echo-time of 0.05 ms. A convolutional neural network was then trained for semantic lung segmentation using 17,713 2D coronal slices, each paired with a label obtained from manual segmentation. Subsequently, the network was applied to 4920 independent 2D test images and results were compared to a manual segmentation using the Sørensen-Dice similarity coefficient (DSC) and the Hausdorff distance (HD). Obtained lung volumes and fractional ventilation values calculated from both segmentations were compared using Pearson's correlation coefficient and Bland Altman analysis. To investigate generalizability to patients outside the CF collective, in particular to those exhibiting larger consolidations inside the lung, the network was additionally applied to UTE images from four patients with pneumonia and one with lung cancer., Results: The overall DSC for lung tissue was 0.967 ± 0.076 (mean ± standard deviation) and HD was 4.1 ± 4.4 mm. Lung volumes derived from manual and deep learning based segmentations as well as values for fractional ventilation exhibited a high overall correlation (Pearson's correlation coefficent = 0.99 and 1.00). For the additional cohort with unseen pathologies / consolidations, mean DSC was 0.930 ± 0.083, HD = 12.9 ± 16.2 mm and the mean difference in lung volume was 0.032 ± 0.048 L., Conclusions: Deep learning-based image segmentation in stack-of-spirals based lung MRI allows for accurate estimation of lung volumes and fractional ventilation values and promises to replace the time-consuming step of manual image segmentation in the future.

Background: Computed tomography (CT) pulmonary angiography is the diagnostic reference standard in suspected pulmonary embolism (PE). Favorable results for dual-energy CT (DECT) images have been reported for this condition. Nowadays, dual-energy data acquisition is feasible with different technical options, including a single-source split-filter approach. Therefore, the aim of this retrospective study was to investigate image quality and radiation dose of thoracic split-filter DECT in comparison to conventional single-energy CT in patients with suspected PE., Methods: A total of 110 CT pulmonary angiographies were accomplished either as standard single-energy CT with automatic tube voltage selection (ATVS) (n=58), or as split-filter DECT (n=52). Objective [pulmonary artery CT attenuation, signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR)] and subjective image quality [four-point Likert scale; three readers (R)] were compared among the two study groups. Size-specific dose estimates (SSDE), dose-length-product (DLP) and volume CT dose index (CTDIvol) were assessed for radiation dose analysis., Results: Split-filter DECT images yielded 67.7% higher SNR (27.0 vs. 16.1; P<0.001) and 61.9% higher CNR (22.5 vs. 13.9; P<0.001) over conventional single-energy images, whereas CT attenuation was significantly lower (344.5 vs. 428.2 HU; P=0.013). Subjective image quality was rated good or excellent in 93.0%/98.3%/77.6% (R1/R2/R3) of the single-energy CT scans, and 84.6%/82.7%/80.8% (R1/R2/R3) of the split-filter DECT scans. SSDE, DLP and CTDIvol were significantly lower for conventional single-energy CT compared to split-filter DECT (all P<0.05), which was associated with 26.7% higher SSDE., Conclusions: In the diagnostic workup of acute PE, the split-filter allows for dual-energy data acquisition from single-source single-layer CT scanners. The existing opportunity to assess pulmonary "perfusion" based on analysis of iodine distribution maps is associated with higher radiation dose in terms of increased SSDE than conventional single-energy CT with ATVS. Moreover, a proportion of up to 3.8% non-diagnostic examinations in the current reference standard test for PE is not negligible., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/qims-20-740). Dr. BP reports personal fees from Siemens Healthcare GmbH and grants from Siemens Healthcare GmbH, outside the submitted work. Dr. AMW reports grants from Siemens Healthcare GmbH, outside the submitted work; Dr. JPG reports personal fees from Siemens Healthcare GmbH, grants from Interdisciplinary Center of Clinical Research Würzburg and grants from Siemens Healthcare GmbH, outside the submitted work; Dr. SV reports grants from Siemens Healthcare GmbH (money payed to the institution), outside the submitted work. Prof. TAB reports grants from Deutsche Forschungsgesellschaft, grants from Siemens Healthcare GmbH, personal fees from Roche/Chugai, personal fees from Novartis, outside the submitted work. The other authors have no conflicts of interest to declare., (2021 Quantitative Imaging in Medicine and Surgery. All rights reserved.)

Background In cystic fibrosis (CF), recurrent imaging and pulmonary function tests (PFTs) are needed for the assessment of lung function during disease management. Purpose To assess the clinical feasibility of pulmonary three-dimensional ultrashort echo time (UTE) MRI at breath holding for quantitative image analysis of ventilation inhomogeneity and hyperinflation in CF compared with PFT. Materials and Methods In this prospective study from May 2018 to June 2019, participants with CF and healthy control participants underwent PFTs and functional lung MRI by using a prototypical single breath-hold three-dimensional UTE sequence. Fractional ventilation (FV) was calculated from acquired data in normal inspiration and normal expiration. FV of each voxel was normalized to the whole lung mean (FV N ), and interquartile range of normalized ventilation (IQR N ; as a measure of ventilation heterogeneity) was calculated. UTE signal intensity (SI) was assessed in full expiration (SI N , normalized to aortic blood). Obtained metrics were compared between participants with CF and control participants. For participants with CF, MRI metrics were correlated with the standard lung clearance index (LCI) and PFT. Mann-Whitney U tests and Spearman correlation were used for statistical analysis. Results Twenty participants with CF (mean age, 17 years ± 9 [standard deviation]; 12 men) and 10 healthy control participants (24 years ± 8; five men) were included. IQR N was higher for participants with CF than for control participants (mean, 0.66 ± 0.16 vs 0.50 ± 0.04, respectively; P = .007). In the 20 participants with CF, IQR N correlated with obstruction markers forced expiratory volume in 1 second-to-forced vital capacity ratio ( r = -0.70; 95% confidence interval [CI]: -0.92, -0.28; P < .001), mean expiratory flow 25% ( r = 0.78; 95% CI: -0.95, -0.39; P < .001), and with the ventilation inhomogeneity parameter LCI ( r = 0.90; 95% CI: 0.69, 0.96; P < .001). Mean SI N in full expiration was lower in participants with CF than in control participants (0.34 ± 0.08 vs 0.39 ± 0.03, respectively; P = .03). Conclusion Three-dimensional ultrashort echo time MRI in the lungs allowed for functional imaging of ventilation inhomogeneity within a few breath holds in patients with cystic fibrosis. © RSNA, 2020 Online supplemental material is available for this article. See also the editorial by Wielpütz in this issue.

Purpose: To evaluate three-dimensional (3D) ultrashort echo time (UTE) MRI regarding image quality and suitability for functional image analysis using gradient-echo sequences in breath-hold and with self-navigation., Materials and Methods: In this prospective exploratory study, 10 patients (mean age, 21 years; age range, 5-58 years; five men) and 10 healthy control participants (mean age, 25 years; age range, 10-39 years; five men) underwent 3D UTE MRI at 3.0 T. Imaging was performed with a prototypical stack-of-spirals 3D UTE sequence during single breath holds (echo time [TE], 0.05 msec) and with a self-navigated "Koosh ball" 3D UTE sequence at free breathing (TE, 0.03 msec). Image quality was rated on a four-point Likert scale. Edge sharpness was calculated. After semiautomated segmentation, fractional ventilation was calculated from MRI signal intensity (FV SI ) and volume change (FV Vol ). The air volume fraction (AVF) was estimated from relative signal intensity (aortic blood signal intensity was used as a reference). Means were compared between techniques and participants. The Wilcoxon signed rank test and Spearman rank correlation were used for statistical analyses., Results: Image quality ratings were equal for both techniques. However, stack-of-spirals breath-hold UTE was more susceptible to motion and aliasing artifacts. Mean FV SI was higher during breath hold than at free breathing (mean ± standard deviation in milliliters of gas per milliliters of parenchyma, 0.17 mL/mL ± 0.06 [minimum, 0.07; maximum, 0.34] vs 0.11 mL/mL ± 0.03 [minimum, 0.06; maximum, 0.17], P = .016). Mean FV SI and FV Vol were in good agreement (mean difference: at breath hold, -0.008 [95% confidence interval {CI}: 0.007, -0.024]; ρ = 0.97 vs free breathing, -0.004 [95% CI: 0.007, -0.016]; ρ = 0.91). AVF correlated between both techniques (ρ = 0.94)., Conclusion: Breath-hold and self-navigated 3D UTE sequences yield proton density-weighted images of the lungs that are similar in quality, and both techniques are suitable for functional image analysis. Supplemental material is available for this article. © RSNA, 2020., Competing Interests: Disclosures of Conflicts of Interest: J.F.H. disclosed no relevant relationships. S.V. disclosed no relevant relationships. C.M. disclosed no relevant relationships. L.M.P. disclosed no relevant relationships. T.B. Activities related to the present article: employed by Siemens Healthcare GmbH. Activities not related to the present article: disclosed no relevant relationships. Other relationships: disclosed no relevant relationships. J.P. Activities related to the present article: disclosed no relevant relationships. Activities not related to the present article: disclosed money paid to author from Siemens Healthcare. Other relationships: disclosed no relevant relationships. T.A.B. Activities related to the present article: disclosed no relevant relationships. Activities not related to the present article: disclosed consultancy fees from Roche/Chugai for membership on the Expert Panel for “Imaging in Giant Cell Arteritis” and from Novartis for in-house training for “Radiology in Oncological Studies;” grant pending from Deutsche Forschungsgemeinschaft for imaging giant cells in arteritis; payment for lectures about imaging in giant cell arteritis from Roche/Chugai. Other relationships: disclosed no relevant relationships. H.K. disclosed no relevant relationships. A.M.W. disclosed no relevant relationships., (2020 by the Radiological Society of North America, Inc.)

Background: To increase the image quality of end-expiratory and end-inspiratory phases of retrospective respiratory self-gated 4D MRI data sets using non-rigid image registration for improved target delineation of moving tumors., Methods: End-expiratory and end-inspiratory phases of volunteer and patient 4D MRI data sets are used as targets for non-rigid image registration of all other phases using two different registration schemes: In the first, all phases are registered directly (dir-Reg) while next neighbors are successively registered until the target is reached in the second (nn-Reg). Resulting data sets are quantitatively compared using diaphragm and tumor sharpness and the coefficient of variation of regions of interest in the lung, liver, and heart. Qualitative assessment of the patient data regarding noise level, tumor delineation, and overall image quality was performed by blinded reading based on a 4 point Likert scale., Results: The median coefficient of variation was lower for both registration schemes compared to the target. Median dir-Reg coefficient of variation of all ROIs was 5.6% lower for expiration and 7.0% lower for inspiration compared with nn-Reg. Statistical significant differences between the two schemes were found in all comparisons. Median sharpness in inspiration is lower compared to expiration sharpness in all cases. Registered data sets were rated better compared to the targets in all categories. Over all categories, mean expiration scores were 2.92 ± 0.18 for the target, 3.19 ± 0.22 for nn-Reg and 3.56 ± 0.14 for dir-Reg and mean inspiration scores 2.25 ± 0.12 for the target, 2.72 ± 215 0.04 for nn-Reg and 3.78 ± 0.04 for dir-Reg., Conclusions: In this work, end-expiratory and inspiratory phases of a 4D MRI data sets are used as targets for non-rigid image registration of all other phases. It is qualitatively and quantitatively shown that image quality of the targets can be significantly enhanced leading to improved target delineation of moving tumors.

Background: Contrast-enhanced cardiovascular magnetic resonance angiography (CE-CMRA) is the established imaging modality for patients with Marfan syndrome requiring life-long annual aortic imaging before and after aortic root replacement. Contrast-free CMRA techniques avoiding side-effects of contrast media are highly desirable for serial imaging but have not been evaluated in the postoperative setup of Marfan patients. The purpose of this study was to assess the feasibility of non-contrast balanced steady-state free precession (bSSFP) magnetic resonance imaging for aortic monitoring of postoperative patients with Marfan syndrome., Methods: Sixty-four adult Marfan patients after aortic root replacement were prospectively included. Fourteen patients (22%) had a residual aortic dissection after surgical treatment of type A dissection. bSSFP imaging and CE-CMRA were performed at 1.5 Tesla. Two radiologists evaluated the images regarding image quality (1 = poor, 4 = excellent), artifacts (1 = severe, 4 = none) and aortic pathologies. Readers measured the aortic diameters at defined levels in both techniques. Statistics included observer agreement for image scoring and diameter measurements and ROC analyses for comparison of the diagnostic performance of bSSFP and CE-CMRA., Results: Both readers observed no significant differences in image quality between bSSFP and CE-CMRA and found a median image quality score of 4 for both techniques (all p > .05). No significant differences were found regarding the frequency of image artifacts in both sequences (all p > .05). Sensitivity and specificity for detection of aortic dissections was 100% for both readers and techniques. Compared to bSSFP imaging, CE-CMRA resulted in higher diameters (mean bias, 0.9 mm; p < .05). The inter-observer biases of diameter measurements were not significantly different (all p > .05), except for the distal graft anastomosis (p = .001). Using both techniques, the readers correctly identified a graft suture dehiscence with aneurysm formation requiring surgery., Conclusion: Unenhanced bSSFP CMR imaging allows for riskless aortic monitoring with high diagnostic accuracy in Marfan patients after aortic root surgery.

Aim: To evaluate the risk profile of sulfur hexafluoride in voiding urosonography (VUS) based on a large cohort of children., Methods: Since 2011 sulfur hexafluoride (SH, SonoVue ® , Bracco, Italy) is the only ultrasound contrast available in the European Union and its use in children has not been approved. Within a 4-year-period, 531 children with suspected or proven vesicoureteral reflux (f/m = 478/53; mean age 4.9 years; 1 mo-25.2 years) following parental informed consent underwent VUS with administration of 2.6 ± 1.2 mL SH in a two-center study. A standardized telephone survey on adverse events was conducted three days later., Results: No acute adverse reactions were observed. The survey revealed subacute, mostly self-limited adverse events in 4.1% (22/531). The majority of observed adverse events (17/22) was not suspected to be caused by an allergic reaction: Five were related to catheter placement, three to reactivated urinary tract infections, five were associated with perineal disinfection before voiding urosonography or perineal dermatitis and four with a common cold. In five patients (0.9%) hints to a potential allergic cause were noted: Perineal urticaria was reported in three interviews and isolated, mild fever in two. These were minor self-limited adverse events with a subacute onset and no hospital admittance was necessary. Ninety-six point two percent of the parents would prefer future VUS examinations with use of SH., Conclusion: No severe adverse events were observed and indications of self-limited minor allergic reactions related to intravesical administration of SH were reported in less than 1%., Competing Interests: Conflict-of-interest statement: Thorsten Bley: Consultancy for GSK and MSD; Speakers bureau: Bayer, Bracco, GE, Guerbet, HeartFlow, Siemens; Research funding: Deutsche Forschungsgesellschaft DFG: BL 1132/1-2; Research cooperation: Siemens, Noras, Rapid. The remaining authors of this manuscript have no conflicts of interest to disclose.

Despite the fact that non-viral nucleic acid delivery systems are generally considered to be less efficient than viral vectors, they have gained much interest in recent years due to their superior safety profile compared to their viral counterpart. Among these synthetic vectors are cationic polymers, branched dendrimers, cationic liposomes and cell-penetrating peptides (CPPs). The latter represent an assortment of fairly unrelated sequences essentially characterised by a high content of basic amino acids and a length of 10-30 residues. CPPs are capable of mediating the cellular uptake of hydrophilic macromolecules like peptides and nucleic acids (e.g. siRNAs, aptamers and antisense-oligonucleotides), which are internalised by cells at a very low rate when applied alone. Up to now, numerous sequences have been reported to show cell-penetrating properties and many of them have been used to successfully transport a variety of different cargos into mammalian cells. In recent years, it has become apparent that endocytosis is a major route of internalisation even though the mechanisms underlying the cellular translocation of CPPs are poorly understood and still subject to controversial discussions. In this review, we will summarise the latest developments in peptide-based cellular delivery of nucleic acid cargos. We will discuss different mechanisms of entry, the intracellular fate of the cargo, correlation studies of uptake versus biological activity of the cargo as well as technical problems and pitfalls.

Cell-penetrating peptides (CPPs) have evolved as promising new tools to deliver nucleic acids into cells. So far, the majority of these delivery systems require a covalent linkage between carrier and cargo. To exploit the higher flexibility of a non-covalent strategy, we focused on the characterisation of a novel carrier peptide termed MPGalpha, which spontaneously forms complexes with nucleic acids. Using a luciferase-targeted small interfering RNA (siRNA) as cargo, we optimised the conditions for MPGalpha-mediated transfection of mammalian cells. In this system, reporter gene activity could be inhibited up to 90% with an IC50 value in the sub-nanomolar range. As a key issue, we addressed the cellular uptake mechanism of MPGalpha/siRNA complexes applying various approaches. First, transfection of HeLa cells with MPGalpha/siRNA complexes in the presence of several inhibitors of endocytosis showed a significant reduction of the RNA interference (RNAi) effect. Second, confocal laser microscopy revealed a punctual intracellular pattern rather than a diffuse distribution of fluorescently labelled RNA-cargo. These data provide strong evidence of an endocytotic pathway contributing significantly to the uptake of MPGalpha/siRNA complexes. Finally, we quantified the intracellular number of siRNA molecules after MPGalpha-mediated transfection. The amount of siRNA required to induce half maximal RNAi was 10 000 molecules per cell. Together, the combination of methods provided allows for a detailed side by side quantitative analysis of cargo internalisation and related biological effects. Thus, the overall efficiency of a given delivery technique as well as the mechanism of uptake can be assessed.