Background:
Many procedures requiring sedation in the pediatric emergency department are performed by consultants from outside the department. This team usually includes orthopedic surgeons and general surgeons. As sedation is now a standard of care in such cases, we evaluated consultants' views on sedation.
Objectives:
To evaluate consultants' views on sedation.
Methods:
A questionnaire with both open-ended questions and Likert-type scores was distributed to all orthopedic surgeons and general surgeons performing procedures during the study period. The questionnaire was presented at three medical centers.
Results:
The questionnaire was completed by 31 orthopedic surgeons and 16 general surgeons. Although the vast majority (93–100%) considered sedation important, a high percentage (64–75%) would still perform such procedures without sedation if not readily available.
Conclusions:
Sedation is very important for patients and although consultants understand its importance, the emergency department staff must be vigilant in both being available and not allowing procedures to "escape" the use of sedation.
Liver cancer is a global health problem, and its incidence is growing worldwide.[1]. Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer (comprising ~75%90% of cases) and is the third most mortal cancer in developing countries with 2-3 times higher rates for men [1-3]. The major risk factors of HCC include chronic viral hepatitis (hepatitis B virus and hepatitis C virus), cirrhosis, chronic alcohol abuse, non-alcoholic fatty liver disease (NAFLD), exposure to aflatoxin, obesity, diabetes, insulin resistance, iron overload, and hypothyroidism[4]. The geographic map of the incidence and mortality of HCC has changed over the last decade and even though most of the cases occur in developing countries, its incidence and mortality in developed countries has increased. This epidemiologic shift is presumed to be a result of the implementation of hepatitis B vaccination and hepatitis C treatment programs worldwide, decreasing the burden of viral hepatitis on liver cancer while increasing the proportion of cases attributed to non-viral risk factors[2, 5-6]. Regardless of the causative agent, early-stage HCC is often asymptomatic leading to delays in timely diagnosis and treatment[7-8]. Owing to the late diagnosis and increasing drug resistance of malignant hepatocytes, treatment of this cancer by conventional chemotherapy agents is challenging. Therefore, development of novel therapeutic approaches aimed at curbing the growth of advanced HCC is highly needed.
Small interfering RNAs (siRNAs) are emerging as attractive therapeutics since it has the potential to reversibly silence any gene with high efficiency and specificity[9, 10]. Yet, siRNA delivery in vivo is still a major challenge due to its rapid degradation by nucleases, poor cellular uptake, and rapid renal clearance following systemic administration[11-13]. In an effort to develop an appropriate delivery system that will overcome these limitations and to improve the safety of potential siRNA -based therapeutics recent research has focused on biocompatible anionic carriers. These carriers have longer circulation time, less interaction with serum components, and lower cytotoxicity compared to cationic carriers.[14-15]. Furthermore, use of delivery systems allows surface modification of the nanoparticles (NPs) and incorporation of targeting ligands that bind specifically to receptors on target cells to induce receptor-mediated endocytosis and improve cellular uptake.
Over the last years, a simple nanoparticulate siRNA delivery platform based on the reversible complexation of siRNA and calcium ions was developed in the. Cohen lab, at the Ben-Gurion University's Department of Biotechnology Engineering. The calcium-siRNA nano-complexes (bearing only a mild negative surface charge) were stable in the presence of serum proteins and successfully induced a very high (~80%) level of gene silencing in several cell types [16]. These promising results led us to develop a novel sulfated polysaccharide-based anionic siRNA delivery platform based on complexation of sulfated polysaccharide (such as alginate sulfate (AlgS)) with siRNA mediated by calcium ions [17]. Incorporating an anionic polymer to the calcium-siRNA nano-complexes can contribute to the stabilization of the NPs and more importantly provide an opportunity to chemically conjugate the polymers with cell-targeting ligands. AIn order to confirm AlgS accessibility on NP surface and ensure maximal accessibility of AlgS for ligand attachment on NPs, we investigated the co-assembly process of the three components, Ca2+, siRNA and AlgS, by applying various component addition strategies and comparing the resulting NPs physico-chemical features (size, zeta potential and surface atomic composition), their intracellular uptake, and gene silencing efficiency in HepG2 (human hepatocellular carcinoma) cells. All of the NPs in this study were prepared in a simple two step self-assembly process and rely on electrostatic interactions bridged by Ca2+[17]. NPs prepared with 5 mM CaCl2, independly of the component addition order, were of average size of 130-150 nm, had a slightly negetive surface charge of < -10mV, were efficiently uptaken by HepG2 cells, and induced approximately ~90% silencing of the Signal transducer and activator of transcription 3 (STAT3) gene () in HepG2 cells. Ca2+ concentrations used in the preparation of these NPs drastically affected the uptake efficiency of AlgS-Ca2+-siRNA NPs. Decreasing [Ca2+] to 2.5 mM completely abolished their cell internalization and STAT3 gene silencing ability.
We confirmed AlgS accessibility on NP (AlgS–Ca2+-siRNA NPs) surface by XPS (X-ray photoelectron spectroscopy) analysis. Since it is well documented that the asialoglycoprotein receptors (ASGPRs) overexpressed on the surfaces of hepatocytes are able to selectivity recognize and bind to N-acetylgalactosamine (GalNAc) it appears that a GalNAc conjugated nano-carrier can provide an attractive approach for siRNA delivery to HCC.[18-20]. Therefore, a GalNAc derivative was conjugated directly onto the surface of NPs. The NPs obtained in this synthesis were denoted GalNAc-NPs. To minimize non-specific uptake mechanism (facilitated by Ca2+ ions) we focused our study on GalNAc-NPs prepared with 2.5 mM Ca2+, which were unable to enter HepG2 cells. Notably, theses GalNAc-NPs were able to achieve efficient transfection and silencing in HEPG2 cells via ASGPR receptor-mediate mechanism (Fig. 1). Importantly a bio-distribution study in healthy BALB/c mice after i.v. injection of GalNAc-NPs containing Cy5-siRNA via the tail vein, confirmed improved targeting of GalNAc-NPs to the liver tissue by a factor of ~3 compared to non-targeted NPs or free siRNA, with lesser renal clearance compared to non-targeted NPs.
Overall, the results obtained so far, indicate that GalNAc-NPs produced in simple reproducible protocols may offer a promising platform for targeted delivery of therapeutic siRNA to HepG2 cells. Moreover, incorporation of sulfated polysaccharide (such as AlgS) to our NPs not only provides protection against siRNA degradation but also enables surface modification and ligand bio-conjugation of different targeting moiety. More generally, as targeted delivery of oligonucleotides to liver hepatocytes using GalNAc conjugates has gained a lot of attention in recent years with several agents already in clinical development, it seems very likely that new oligonucleotides could be one of the main feasible approaches for HCC therapy in the future.
Figure 1 Schematic illustration showing cellular intracellular delivery of GalNAc-NPs into HepG2 cells via ligand-receptor (ASGP) recognition which are overexpressed on their surfaces.
References
1. Llovet JM, Kelley RK, Villanueva A, et al. Hepatocellular carcinoma. Nat Rev Dis Primers. 2021;7(1):6.
2. Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209-49.
3. Rawla P, Sunkara T, Muralidharan P, Raj JP. Update in global trends and aetiology of hepatocellular carcinoma. Contemp Oncol (Pozn). 2018;22(3):141-50.
4. Petruzziello A. Epidemiology of Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) Related Hepatocellular Carcinoma. Open Virol J. 2018;12:26-32.
5. Singal AG, Lampertico P, Nahon P. Epidemiology and surveillance for hepatocellular carcinoma: New trends. J Hepatol. 2020;72(2):250-61.
6. Lurje I, Czigany Z, Bednarsch J, et al. Treatment Strategies for Hepatocellular Carcinoma ⁻ a Multidisciplinary Approach. Int J Mol Sci. 2019;20(6):1465.
7. Draper A. A concise review of the changing landscape of hepatocellular carcinoma. Am J Manag Care. 2020;26(10 Suppl):S211-19.
8. Che L, Paliogiannis P, Cigliano A, Pilo MG, Chen X, Calvisi DF. Pathogenetic, Prognostic, and Therapeutic Role of Fatty Acid Synthase in Human Hepatocellular Carcinoma. Front Oncol. 2019;9:1412.
9. Miele E, Spinelli GP, Miele E, et al. Nanoparticle-based delivery of small interfering RNA: challenges for cancer therapy. Int J Nanomedicine. 2012;7:3637-57.
10. Lee JM, Yoon TJ, Cho YS. Recent developments in nanoparticle-based siRNA delivery for cancer therapy. Biomed Res Int. 2013; 782041.
11. Deng Y, Wang CC, Choy KW, et al. Therapeutic potentials of gene silencing by RNA interference: principles, challenges, and new strategies. Gene. 2014;538(2):217-27.
12. Hong CA, Nam YS. Functional nanostructures for effective delivery of small interfering RNA therapeutics. Theranostics. 2014 ;4(12):1211-32.
13. Ozpolat B, Sood AK, Lopez-Berestein G. Nanomedicine based approaches for the delivery of siRNA in cancer. J Intern Med. 2010;267(1):44-53.
14. Zeng X, de Groot AM, Sijts AJ, et al. Surface coating of siRNA-peptidomimetic nano-self-assemblies with anionic lipid bilayers: enhanced gene silencing and reduced adverse effects in vitro. Nanoscale. 2015;7(46):19687-98.
15. Xie Y, Qiao H, Su Z, Chen M, Ping Q, Sun M. PEGylated carboxymethyl chitosan/calcium phosphate hybrid anionic nanoparticles mediated hTERT siRNA delivery for anticancer therapy. Biomaterials. 2014;35(27):7978-91.
16. Ruvinov E, Kryukov O, Forti E, Korin E, Goldstein M, Cohen S. Calcium-siRNA nanocomplexes: what reversibility is all about. J Control Release. 2015;203:150-60.
17. Korin E, Bejerano T, Cohen S. GalNAc bio-functionalization of nanoparticles assembled by electrostatic interactions improves siRNA targeting to the liver. J Control Release. 2017;266:310-20.
18. Khorev O, Stokmaier D, Schwardt O, Cutting B, Ernst B. Trivalent, Gal/GalNAc-containing ligands designed for the asialoglycoprotein receptor. Bioorg Med Chem. 2008;16(9):5216-31.
19. D'Souza AA, Devarajan PV. Asialoglycoprotein receptor mediated hepatocyte targeting - strategies and applications. J Control Release. 2015;203:126-39.
20. Medina SH, Tekumalla V, Chevliakov MV, Shewach DS, Ensminger WD, El-Sayed ME. N-acetylgalactosamine-functionalized dendrimers as hepatic cancer cell-targeted carriers. Biomaterials. 2011;32(17):4118-29.