Nanoparticles have facilitated unprecedented study of biological processes and molecular markers within a variety of cell samples (reviewed in [1–4]). Diagnostic assays where nanoparticles are used to detect the presence and/or absence of a combination of cell markers are becoming increasingly significant in the identification of progenitor or stem-like cells found within a variety of tumors . While nanotechnology has pioneered major advances in cancer detection, diagnosis, and treatment , tumors within brain continue to pose one of the lowest survival rates five years after diagnosis . While such poor prognosis is largely associated with the highly invasive nature of malignant brain tumors [8–10], the cellular heterogeneity of diseased brain also plays a large role, as constituent subpopulations of neoplastic cells with stem-like properties  appear to be resistant to conventional radiotherapy and chemotherapeutic regimens . Emerging studies have underscored the significance of intracellular markers when identifying neoplastic stem-like populations (reviewed in ), either in tandem with existing extracellular markers (e.g. CD133, PAX6, reviewed in ) or alone. Numerous cytosolic molecules currently serve as therapeutic targets for radiosensitization, including heat shock proteins , binding proteins , Hypoxia Inducible Factors HIF1 and HIF2 , transcription factors , and phospholipoases . In addition, recent studies point to cytosolic markers as excellent detectors of biochemical signatures from cells previously thought to evade the neural system, such as the prion-like protein Doppel (Dpl) found in the male reproductive system , and light neurofilament proteins and class III β-tubulin found in bone marrow-derived mesenchymanl stem cells .
Labeling of intracellular molecules is notoriously difficult to achieve using nanoparticles because of the highly esoteric selectivity required . Intracellular delivery of nanoparticles is strongly affected by both the nature of the particle and the type of cell examined (reviewed in ). For example, established delivery methods of bioconjugates, such as Quantum dots (Qdots), via endocytosis, pinocytosis and injection are known to alter cell function as well as exhibit varied effectiveness per cell type and/or experimental condition [24, 25]. Further, alternative approaches such as electroporation , nanoneedles , and cell-penetrating peptides  have led to internalized Qdots that can become trapped within the endocytic pathway and/or form large aggregates in the cytoplasm . Most recently, researchers have utilized cell penetrating peptides [30, 31], pH-dependent fusogenic peptides , as well as logic-embedded vectors  to achieve endosomal release after internalization. Others have minimized endosomal trapping by using silica , gold [35, 36], and polymer-based nanoparticles  and polyactic acid , while yet others have disrupted endocytosis by using light-activated disruption of intracellular vesicles , or controlled sub-cellular damage of endosomal structures .
Recent applications have revived the practice of nanoparticle encapsulation by incorporating nanoparticles within patented synthetic proteins and polymers, as well as within antiretroviral complexes , each with a varying degree of endosomal escape. Our group has previously shown that cationic liposomes are able to facilitate intracellular delivery of Qdots within live brain cancer cells , but demonstrated that the method is cell line-dependent: Liposomal delivery of Qdots was cytoplasmic within glioblastoma-derived cells, but resulted in endocytosis and trapping of liposomes within endosomes when HeLa cells were used. More unconventional approaches to nanoparticle delivery have begun to incorporate viruses previously used to deliver other nanosized molecules, such as DNA, synthetic oligonucleotides, and pharmaceuticals . Chymeric bacteriophages have been employed to target tumors and introduce intracellular agents bound to its surface , while the plant mosaic virus  was used to incorporate Qdots coated with various molecules (e.g. streptavidin-biotin, dihydrolipoic acid) within its capsid. A recent study adapted the simian virus 40 capsid to encapsulate Qdots functionalized with different surface coatings (e.g. DNA, PEG) for transport within kidney cells . While delivery was successful, it remained unclear whether the virus itself enabled cytosolic release of Qdots or if the Qdots remained trapped within cellular compartments .
The current study has achieved cytoplasmic delivery of targeted Qdots via chimeric fusions between the Sendai virus and cationic liposomes . The Sendai virus is a mouse parainfluenza virus that has been safely used for over two decades, in vitro and in vivo, to deliver molecules such as plasmid DNAs, siRNAs, proteins, iron particles, and pharmaceuticals into numerous cell types (reviewed in ). Its role as a delivery vector capitalizes on two types of proteins present in its capsid: (i) Hemagglutinating and Neuraminidase (HN) proteins, used for attachment of the virus to neuraminic acid-containing receptors on host cells, hemagglutination of erythrocytes, and neuraminidase activity ; and (ii) Fusion (F) proteins needed for virus penetration of host cell membranes, virus-induced hemolysis, and cell fusion [49–51]. In this work we use the Sendai virus to generate virus-based liposomes that achieve cytosolic delivery of targeted Qdots into live Human brain tumor cells with high, consistent efficiency. Qdots were functionalized with a monoclonal biotinylated antibody (Ab) designed to specifically recognize an intracellular epitope of the Epidermal Growth Factor Receptor (EGFR). EGFR was chosen as a candidate target protein because its over-expression and up-regulation is recognized as a significant step in the pathogenesis and progression of a wide variety of cancers, including tumors of the brain [52–55]. Further, previous work from our laboratory has successfully labeled activated EGFR populations in live brain cancer cells by binding Qdots to the extracellular domain of EGFR and then inducing receptor activation to detect intracellular, activated EGFR . In the current study, delivery of Qdot by chimeric fusions between the Sendai virus and cationic liposomes, henceforth called virus-based liposomes (VBLs), was assessed using three different Human cancer cell types: (i) Medulloblastoma (MB), the most common form of pediatric brain tumor; (ii) Glioblastoma (GBM), the most common form of tumor in adult brain; and (iii) HeLa cervical cancer, a well-studied cell line used here as an experimental control.