Sustained hydrogel-based delivery of RNA interference nanocomplexes for gene knockdown

Scaffold based delivery of RNA interference (RNAi) molecules such as free small interfering RNA (siRNA) and microRNA has recently begun to be employed towards treatment of diseases such as cancer, bone regeneration, muscular dystrophy and cardiovascular disease. Effective translation from bench side to clinical use of RNAi has been limited in part because upon systemic delivery the RNAi molecules are degraded by RNases and flushed by excretory organs causing an inefficient duration of gene silencing effect at target tissues. These challenges can potentially be minimised by delivering RNAi molecules via non-viral nanoparticle carriers encapsulated in biocompatible, biodegradable and injectable scaffolds such as hydrogels. Various scaffolds have been shown to aid in sustained localised delivery of RNAi molecules and improve gene silencing. This research focused on optimising and establishing such an RNAi hydrogel-siRNA-nanoparticle (hydrogel-nanocomplex) system for targeted and sustained gene knockdown both in vitro and in vivo using dendrimer and lipid based nanoparticles in combination with synthetic polyethylene glycol (PEG) and natural fibrin hydrogel scaffolds. Four siRNA nanocarriers were investigated for siRNA delivery, that is, fourth generation dendrimer nanoparticles poly(amidoamine) (D) and its modified version (MD) with PEG and a lipid 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) molecule, commercial lipid based Lipofectamine® RNAiMax and Invivofectamine® 3.0 nanoparticles. D and MD achieved better RNase protection compared to lipid nanocomplexes though Invivofectamine® 3.0 nanocomplexes protected a small percentage of siRNA over 10 days. The MD nanoparticle displayed improved siRNA release and transfection efficacy compared to D but efficacy of the dendrimers was lower than the lipid particles. Four hydrogels that have not been investigated for RNAi were assessed for sustainability. Namely, hydrolytically and proteolytically degradable PEG-acrylate (PEGAC), proteolytically degradable PEG - vinyl sulfone (PEG-VS) hydrogels, unmodified fibrin and PEGylated fibrin hydrogel. The nanocomplex release rate in vitro from the various hydrogels showed minimal release from PEGylated hydrogels, burst release from unmodified fibrin and sustained release from PEGylated fibrin. Invivofectamine® 3.0 nanocomplexes retained efficacy optimally after release from PEGylated fibrin hence this hydrogel was utilised for downstream analysis. For in vivo sustained delivery to be effective, determination of hydrogel persistence in vivo was required. After injection in the mouse tibialis anterior (TA) muscle PEG-AC and PEGylated fibrin gels degraded within 2 days. The efficacy of the various nanocomplexes was assayed in a 3D assay that more closely resembled delivery in soft tissue. PEGylated fibrin containing nanocomplexes with cell death siRNA sequences was polymerised around a preformed PEGylated fibrin cell containing droplet. Invivofectamine® 3.0 nanocomplex consistently achieved the highest gene knockdown effect with no evidence of cytotoxicity whilst Lipofectamine® RNAiMax was ineffective. MD showed signs of cytotoxicity when delivered in a sustained fashion. Thus Invivofectamine® 3.0 nanocomplexes in PEGylated fibrin hydrogel were found to be the optimal gel-nanocomplex system to proceed to in vivo assessment. BALB/c GFP transgenic injected in their TA muscle with Invivofectamine® 3.0 nanocomplexes made with siRNA targeting GFP or myostatin (siGFP/siMSTN) in the presence or absence of PEGylated fibrin gel were analysed 7 days post treatment for siRNA retention and GFP and Mstn gene knockdown. Increased retention of siRNA after encapsulation in PEGylated fibrin was observed at 7 days. A non-significant reduction in GFP protein was seen for limbs injected with siGFP- fibrin after 7 days. A substantial and significant reduction in Mstn mRNA levels was elicited by delivery of siMstn–fibrin. Furthermore, only siMstn-fibrin resulted in significant increase in muscle mass. In this study, dendrimer based nanoparticles were found to effectively protect siRNA against RNases however lipid based nanocomplexes were the most efficacious at gene knockdown. The combination of Invivofectamine® 3.0 and PEGylated fibrin was shown to be the most effective in 3D assays and as an injectable controlled release scaffold into soft tissue suggesting that this approach has therapeutic potential

Overexpression of Kpnβ1 and Kpnα2 Importin Proteins in Cancer Derives from Deregulated E2F Activity

The Karyopherin superfamily comprises nuclear transport proteins, involved in the shuttling of certain cargo proteins into and out of the nucleus. Karyopherin β1 (Kpnβ1) and Karyopherin α2 (Kpnα2) are importin proteins, which work in concert to transport their cargo into the nucleus. We previously identified increased expression of Kpnβ1 and Kpnα2 in cervical tumours compared to normal epithelium and in transformed cells compared to their normal counterparts. This study therefore aimed to identify the transcription regulatory mechanisms associated with high Kpnβ1 and Kpnα2 levels in cancer cells. Kpnβ1 (−2013 to +100) and Kpnα2 (−1900 to +69) promoter fragments were separately cloned into the reporter vector, pGL3-basic, and luciferase assays revealed both as significantly more active in cancer and transformed cells compared to normal. A series of deletion constructs identified the −637 to −271 Kpnβ1 and −180 to −24 Kpnα2 promoter regions as responsible for the differential promoter activity, and a number of highly conserved E2F binding sites were identified within these regions. Mutation analysis confirmed the requirement of E2F sites for promoter activity, and ChIP analysis confirmed E2F2/Dp1 binding to the Kpnβ1 and Kpnα2 promoters in vivo. Dp1 inhibition resulted in decreased levels of the respective proteins, confirming the role of E2F in the overexpression of Kpnβ1 and Kpnα2 proteins in cancer. E2F activity is known to be deregulated in cervical cancer cells due to the inhibition of its repressor, Rb, by HPV E7. The inhibition of E7 using siRNA resulted in decreased Kpnβ1 and Kpnα2 promoter activities, as did the overexpression of Rb. In conclusion, this study is a first to show that elevated Kpnβ1 and Kpnα2 expression in cancer cells correlates with altered transcriptional regulation associated with deregulated E2F/Rb activitie

The garlic compound ajoene covalently binds vimentin, disrupts the vimentin network and exerts anti-metastatic activity in cancer cells

Background Garlic has been used for centuries for its flavour and health promoting properties that include protection against cancer. The vinyl disulfide-sulfoxide ajoene is one of the phytochemicals found in crushed cloves, hypothesised to act by S-thiolating reactive cysteines in target proteins. Methods Using our fluorescently labelled ajoene analogue called dansyl-ajoene, ajoene’s protein targets in MDA-MB-231 breast cancer cells were tagged and separated by 2D electrophoresis. A predominant band was identified by MALDI-TOF MS/MS to be vimentin. Target validation experiments were performed using pure recombinant vimentin protein. Computational modelling of vimentin bound to ajoene was performed using Schrödinger and pKa calculations by Epik software. Cytotoxicity of ajoene in MDA-MB-231 and HeLa cells was measured by the MTT assay. The vimentin filament network was visualised in ajoene-treated and non-treated cells by immunofluorescence and vimentin protein expression was determined by immunoblot. The invasion and migration activity was measured by wound healing and transwell assays using wildtype cells and cells in which the vimentin protein had been transiently knocked down by siRNA or overexpressed. Results The dominant protein tagged by dansyl-ajoene was identified to be the 57 kDa protein vimentin. The vimentin target was validated to reveal that ajoene and dansyl-ajoene covalently bind to recombinant vimentin via a disulfide linkage at Cys-328. Computational modelling showed Cys-328 to be exposed at the termini of the vimentin tetramer. Treatment of MDA-MB-231 or HeLa cells with a non-cytotoxic concentration of ajoene caused the vimentin filament network to condense; and to increase vimentin protein expression. Ajoene inhibited the invasion and migration of both cancer cell lines which was found to be dependent on the presence of vimentin. Vimentin overexpression caused cells to become more migratory, an effect that was completely rescued by ajoene. Conclusions The garlic-derived phytochemical ajoene targets and covalently modifies vimentin in cancer cells by S-thiolating Cys-328. This interaction results in the disruption of the vimentin filament network and contributes to the anti-metastatic activity of ajoene in cancer cells

Prevalence of Myocardial Injury and Myocardial Infarction in Patients with a Hypertensive Emergency: A Systematic Review

Myocardial injury and myocardial infarction can complicate a hypertensive emergency, and both are associated with poor prognosis. However, little is known about the prevalence of myocardial injury and the different subtypes of myocardial infarction in patients with hypertensive emergencies. This systematic review aims to determine the prevalence of myocardial infarction and its subtypes, and the prevalence of myocardial injury in patients with hypertensive emergencies following the PRISMA guideline. A systematic search of PubMed, Web of Science, and EBSCOHost (MEDLINE) databases was carried out from inception to identify relevant articles. A total of 18 studies involving 7545 patients with a hypertensive emergency were included. Fifteen (83.3%) studies reported on the prevalence of myocardial infarction ranging from 3.6% to 59.6%, but only two studies specifically indicated the prevalence of ST-elevation and non-ST-elevation myocardial infarction. The prevalence of myocardial injury was obtained in three studies (16.7%) and ranged from 15% to 63%. Despite being common, very few studies reported myocardial injury and the subtypes of myocardial infarction among patients presenting with a hypertensive emergency, highlighting the need for more research in this area which will provide pertinent data to guide patient management and identify those at increased risk of major adverse cardiovascular events

Prevalence of Myocardial Injury and Myocardial Infarction in Patients with a Hypertensive Emergency: A Systematic Review

Myocardial injury and myocardial infarction can complicate a hypertensive emergency, and both are associated with poor prognosis. However, little is known about the prevalence of myocardial injury and the different subtypes of myocardial infarction in patients with hypertensive emergencies. This systematic review aims to determine the prevalence of myocardial infarction and its subtypes, and the prevalence of myocardial injury in patients with hypertensive emergencies following the PRISMA guideline. A systematic search of PubMed, Web of Science, and EBSCOHost (MEDLINE) databases was carried out from inception to identify relevant articles. A total of 18 studies involving 7545 patients with a hypertensive emergency were included. Fifteen (83.3%) studies reported on the prevalence of myocardial infarction ranging from 3.6% to 59.6%, but only two studies specifically indicated the prevalence of ST-elevation and non-ST-elevation myocardial infarction. The prevalence of myocardial injury was obtained in three studies (16.7%) and ranged from 15% to 63%. Despite being common, very few studies reported myocardial injury and the subtypes of myocardial infarction among patients presenting with a hypertensive emergency, highlighting the need for more research in this area which will provide pertinent data to guide patient management and identify those at increased risk of major adverse cardiovascular events

Cardiac Complications of Hypertensive Emergency: Classification, Diagnosis and Management Challenges

While mortality in patients with hypertensive emergency has significantly improved over the past decades, the incidence and complications associated with acute hypertension-mediated organ damage have not followed a similar trend. Hypertensive emergency is characterized by an abrupt surge in blood pressure, mostly occurring in people with pre-existing hypertension to result in acute hypertension-mediated organ damage. Acute hypertension-mediated organ damage commonly affects the cardiovascular system, and present as acute heart failure, myocardial infarction, and less commonly, acute aortic syndrome. Elevated cardiac troponin with or without myocardial infarction is one of the major determinants of outcome in hypertensive emergency. Despite being an established entity distinct from myocardial infarction, myocardial injury has not been systematically studied in hypertensive emergency. The current guidelines on the evaluation and management of hypertensive emergencies limit the cardiac troponin assay to patients presenting with features of myocardial ischemia and acute coronary syndrome, resulting in underdiagnosis, especially of atypical myocardial infarction. In this narrative review, we aimed to give an overview of the epidemiology and pathophysiology of hypertensive emergencies, highlight challenges in the evaluation, classification, and treatment of hypertensive emergency, and propose an algorithm for the evaluation and classification of cardiac acute hypertension-mediated organ damage

Overexpression of Kpnb1 and Kpna2 Importin Proteins in Cancer Derives from Deregulated E2F Activity

Abstract The Karyopherin superfamily comprises nuclear transport proteins, involved in the shuttling of certain cargo proteins into and out of the nucleus. Karyopherin b1 (Kpnb1) and Karyopherin a2 (Kpna2) are importin proteins, which work in concert to transport their cargo into the nucleus. We previously identified increased expression of Kpnb1 and Kpna2 in cervical tumours compared to normal epithelium and in transformed cells compared to their normal counterparts. This study therefore aimed to identify the transcription regulatory mechanisms associated with high Kpnb1 and Kpna2 levels in cancer cells. Kpnb1 (22013 to +100) and Kpna2 (21900 to +69) promoter fragments were separately cloned into the reporter vector, pGL3-basic, and luciferase assays revealed both as significantly more active in cancer and transformed cells compared to normal. A series of deletion constructs identified the 2637 to 2271 Kpnb1 and 2180 to 224 Kpna2 promoter regions as responsible for the differential promoter activity, and a number of highly conserved E2F binding sites were identified within these regions. Mutation analysis confirmed the requirement of E2F sites for promoter activity, and ChIP analysis confirmed E2F2/Dp1 binding to the Kpnb1 and Kpna2 promoters in vivo. Dp1 inhibition resulted in decreased levels of the respective proteins, confirming the role of E2F in the overexpression of Kpnb1 and Kpna2 proteins in cancer. E2F activity is known to be deregulated in cervical cancer cells due to the inhibition of its repressor, Rb, by HPV E7. The inhibition of E7 using siRNA resulted in decreased Kpnb1 and Kpna2 promoter activities, as did the overexpression of Rb. In conclusion, this study is a first to show that elevated Kpnb1 and Kpna2 expression in cancer cells correlates with altered transcriptional regulation associated with deregulated E2F/Rb activities Citation: van der Watt PJ, Ngarande E, Leaner VD (2011) Overexpression of Kpnb1 and Kpna2 Importin Proteins in Cancer Derives from Deregulated E2F Activity. PLoS ONE 6(11): e27723

Kpnβ1 and Kpnα2 protein levels in normal, transformed and cancer cells correlate with respective promoter activities.

<p>A. Western Blot analysis of Kpnβ1 and Kpnα2 protein levels in normal WI38, transformed SVWI38, and cervical cancer CaSki cells reveals increased expression in the cancer and transformed cells. B, C. −2013 to +100 Kpnβ1 promoter activity (B) and −1900 to +69 Kpnα2 promoter activity (C) was measured in cell lysates prepared from transfected WI38, SVWI38 and CaSki cells, and was observed to be significantly higher in the transformed and cancer cells compared to the normal cells (*p<0.05). TK-Renilla-Luc was used as a control for transfection efficiency and luciferase activity is expressed relative to Renilla luciferase. Results shown are the mean ± SD of experiments performed in triplicate and repeated at least two times.</p

HPV E7 inhibition and Rb overexpression affects the activity of the Kpnβ1 and Kpnα2 promoters.

<p>A, B. HPV16 E7 siRNA was used to inhibit E7 expression in CaSki cells and Western blot analysis performed to analyse HPV E7 (A) and Rb (B) levels after HPV E7 knock-down. C, D. Kpnβ1 (C) and Kpnα2 (D) promoter activities were measured after E7 siRNA transfection, and were found to be significantly inhibited after HPV E7 inhibition. E, F. Rb was overexpressed in CaSki cells and Kpnβ1 (E) and Kpnα2 (F) promoter activities were found to be significantly inhibited. Results shown are the mean ± SE of experiments in quadruplicate performed at least two times (* p<0.05).</p

E2F/DP1 binds and activates the Kpnβ1 and Kpnα2 promoters <i>in vivo</i>.

<p>A, B. Samples of sonicated chromatin were immunoprecipitated with an α-E2F2 antibody, an α-DP1 antibody, or no antibody, as indicated. DNA isolated from immunoprecipitated material was amplified by PCR using primers spanning the E2F sites present in the Kpnβ1 (A) or Kpnα2 (B) promoters. Amplified fragments were analysed by electrophoresis on a 2% agarose gel. Experiments were performed in both SVWI38 and CaSki cell lines. C. Western Blot showing Kpnβ1 and Kpnα2 protein levels after Dp1 inhibition using siRNA. β-tubulin was used as a control for protein loading. D, E. Kpnβ1 and Kpnα2 promoter activities after Dp1 inhibition. Results shown are the mean ± SE of experiments in quadruplicate performed at least two times (* p<0.05).</p