Using large molecules has been more successful: therapeutic anti-E6 gene product approaches, including ribozymes, siRNA, and antibodies have been highly effective in cell culture and animal models [17]C[21]. Anti-E6, large molecule therapeutics require crossing cell membranes to be effective against HPV-induced cancers. cells and remains active. This delivery Edicotinib method is targeted, non-cytotoxic, and non-invasive, making it more easily translatable for experiments than other transfection methods. Introduction Virtually all cervical cancers are dependent on persistent infection by high-risk human papillomavirus (HPV) [1]. Papillomaviruses are also implicated in almost 90% of other anogenital cancers [2]. In addition, oral cancer and non-melanoma skin cancer have an etiological association with high-risk HPVs [3]. Reliable screening procedures exist for cervical cancer, notably the Pap smear. However, cervical cancer still remains prevalent, particularly in populations with reduced access to screening, due to geographical or cultural limitations [4]. Cervical cancer commonly affects women in their thirties and Edicotinib forties [4], significantly impacting the quality of life during their active, younger years. The current treatment for cervical cancer, consisting of cisplatin/radiotherapy combined with surgery, has remained unchanged for the past several years despite its many detrimental side effects, including nausea, fatigue, and toxicity in unaffected organs. In addition, surgical excision of cervical cancerous tissue is a highly invasive procedure, and thus impractical. A more targeted therapy for cervical cancer would help decrease treatment-associated morbidity and overall mortality, and can also be applied to other HPV-related cancers, such as head and neck cancers, the incidence of which is currently on the rise [5]. HPV16 is the most common high-risk papillomavirus type, and like other tumourigenic DNA viruses, encodes viral oncoproteins that act synergistically [6]. Two intracellular oncoproteins, E6 and E7, play an important role in the malignant transformation of HPV-infected cells [6]. E7 induces increased cellular proliferation by binding to and inactivating the tumour suppressor retinoblastoma protein, thereby releasing a transcription factor (E2F) and allowing the HPV-infected cell to proceed through the cell cycle, even in the absence of growth factors [7]. E6 is the main player in cellular immortalization and transformation as well as in upholding tumour growth [8]. These activities are mediated by E6-dependent degradation of cellular proteins (reviewed in [9]) such as the tumour suppressor protein p53 [10] and by promoting telomerase activity [11]. Since E6 is crucial for cervical carcinogenesis and most importantly for maintenance of the malignant phenotype [12], [13], this molecule is an attractive target for new treatment strategies. Initially, small molecule approaches were tried. A library screen of small molecules identified zinc-finger ejecting compounds targeting E6 [14], [15]. However, these compounds have not had the anticipated effect [16] or required excessively high doses to be clinically relevant [15]. Thus, the rational design of small molecules as therapeutic agents that target specific proteins is extremely challenging due to the complex energetics associated with small molecule-protein interactions. Using large molecules has been more successful: therapeutic anti-E6 gene product approaches, including ribozymes, siRNA, and antibodies have been highly effective in cell culture and animal models [17]C[21]. Anti-E6, large molecule therapeutics require crossing cell membranes to be effective against HPV-induced cancers. Chemical transfection reagents are an easy solution to this problem and in clinical environments. A variety of other methods to facilitate cell membrane crossing, including the use of membrane translocating signal transport peptides, electroporation, and even red cell ghosts [22]C[24], have been explored, but again lack ease of translation. Ideally, localized excitation of the membrane that results in transient increased permeability would be well-suited for a clinical application. Such an excitation can be produced by ultrasound, and indeed, high intensity focused ultrasound (HIFU) combined with microbubbles Edicotinib (lipid shell-encased octafluoropropane Edicotinib gas contrast agents), a process known as sonoporation, has been used for ultrasound-mediated intracellular delivery of a variety of molecules such as dextrans, calcein, plasmid DNA, siRNA, and antibodies (Table 1) [25]C[34]. Mechanistic studies Lysipressin Acetate have implied plasma membrane sonoporation as the dominant mechanism underlying ultrasound-enhanced molecule transfer [35]. Reversible pore formation, approximately 100 nm in effective diameter with a half-life of a few seconds, is thought to result from mechanical stress to the cell membrane caused by oscillation and cavitation of the microbubbles under the influence of the acoustic beam [35]. The formation of these pores has been studied using techniques such as: atomic force microscopy; high-speed camera, real-time optical observations of cell/bubble interactions; scanning electron microscopy; and measurement of changes in trans-membrane current [31], [36]C[38]. Microbubbles are routinely used today as an intravenously injected diagnostic drug for contrast enhancement during echocardiographic procedures. Table 1 Examples of experiments using sonoporation to transfer large molecules across the cell membrane. protocols, and potentially, even clinical trial-based experiments, thus filling the gap in translational research Edicotinib that these other methods were unable to address. The feasibility of monoclonal antibody delivery.