Effective treatment of brain tumor remains a challenge in medical science. Gliomas are the most common type of tumors of brain and central nervous system [Giakoumettis D et al., 2018]. Based on the type of primary cells along with molecular characteristics, gliomas can be of astrocytomas, ependymomas, oligodendrogliomas etc. Glioma is characterized by its uncontrolled cellular proliferation, diffused infiltration along with significant angiogenesis (Kesari S et al., 2011). Glioma at its fourth stage is referred to as glioblastoma multiforme, which is the most dangerous stage with poor prognosis and an average survival rate of 1-2 years [Louis DN et al., 2016]. In spite of all the advanced medical strategies, death rate of glioma patients is increasing at an alarming rate all over the world. The treatment failures may be attributed to the delicate and sensitive characteristics of brain tissue, which limits effective application of surgery or radiation therapy; whereas presence of blood-brain barrier (BBB) further worsens the case [Stamatovic SM et al., 2008]. BBB is the most complex, tight endothelial barrier, which strictly checks the entry of therapeutic molecules into the brain and thus stands as a serious challenge for chemotherapy [Guo J et al., 2017; Bhowmik A et al., 2015]. Although, many conventional anticancer drugs are available in clinical practice, but majority of them fails to maintain the desired therapeutic concentration in the brain tissue for a sufficient period of time due to their inability to pass effectively through BBB [Jain KK et al., 2012] Some lipophilic drugs like carmustine, temozolomide, bevacizumab etc. are being claimed to cross BBB, but shorter half-life along with severe dose related toxic effects associated with them throw additional challenges to get desired treatment outcomes (Athmakur H et al., 2018, Chamberlain MC et al., 2010). In this context, novel drug delivery strategies like nanoliposomes, nanoparticles, polymeric micelles, niosomes, dendrimers etc. have been investigated widely in past years to improve the efficacy of conventional chemotherapeutic agents for the treatment of glioma [Hao Y et al., 2015; Li X et al., 2017; Hu X et al., 2017]. However, till today, very few of them have been approved to be used in clinical practice. Among various types of nanocarrier platforms, nanosize lipid based vesicular carriers have been largely preferred for successful delivery of toxic chemotherapeutic drugs to brain [Bondi ML et al., 2012; Laouini A et al., 2012]. Due to high lipophilic nature as well as ultra small size, they fulfill the prime requisite criteria to overcome BBB to get into the brain. Phospholipid based nanostructures (NLs) are the ultra-micron size phospholipid vesicles consisting of self assembled lipid bilayers enclosing small aqueous phase in their core [Akbarzadeh A et al., 2013]. Due to this architectural uniqueness, they act as dual platform for both hydrophobic and hydrophilic molecules. The hydrophobic/lipophilic agents get entrapped in the outer lipid bilayer, where as the hydrophilic agents remain encapsulated in the aqueous core [Akbarzadeh A et al., 2013]. NLs owing to their lipophilicity, biodegradability, non-immunogenicity, biocompatibility, sustained drug release property, ease of surface manipulation etc. have drawn the attention of formulation scientists as preferred drug delivery vehicles in nanomedicine based research [Sonali S et al., 2016; Shufeng Y et al., 2019]. Due to sustained delivery of the loaded cargo as well as site-specific delivery, the dose of the cytotoxic anti-cancer drugs is expected to be reduced, which leading to better treatment outcome and fewer side effects. Lomustine is a nitrosourea class of antineoplastic agent, which is used in the treatment of various types of malignancies, including glioma [Harvey KA et al., 2015]. It inhibits protein synthesis by causing alkylation and cross-linking in the nucleic acids (DNA/RNA). Being lipophlic in nature, it posses the capacity to cross BBB, however, its short half life and deadly side effects like severe bone marrow depression, leucopenia, etc. limits its effective use in the treatment of glioma [Lonardi S et al., 2005; Fisusi FA et al., 2015]. Thus, there is a need to develop novel strategies for the safer and effective delivery of lomustine to brain and thereby reducing the dose-related side effects associated with the conventional forms. Kevin A. Harvey et al. studied anticancer properties of lomustine in conjunction with docosahexaenoic acid (DHA) in glioblastoma cell lines. They studied effects of lomustine, alone and in combination with DHA inC6 human glioblastoma cell line. (Kevin A. Harvey et al., 2015). In another study, lomustine nanoparticles prepared by molecular envelope technology was tested on C6 glioblastoma bearing animal model (Funmilola A. Fisusi et al., 2016). Another work reported an optimized method of development of poly (d,l-lactide-co-glycolide) based lomustine nanoparticles and investigated their anticancer potential in lung cancer cell line L132 (Mehrotra1 A. et al., 2015). However, to our knowledge, no reports are available on the anticancer potential of lomustine loaded lipid nanostructures (LNLs) on C6 glioma cells and also on their in vivo pharmacokinetic (PK) profile. In the lieu of which, the present study aims to investigate the anticancer potential of optimized LNLs on rat glioma cells along with evaluation of both blood and brain PK profiles in experimental animal model. The LNLs will be prepared by conventional method with optimization of critical manufacturing conditions to achieve the desired nanosize. Preferably, we want to keep the size of LNLs within 100 nm range for effective permeation into brain as well as to escape from reticulo-endothelial system. The experimental LNLs will be evaluated by different in vitro techniques and the optimized formulation will be tested for its in vitro anticancer effectiveness in C6 glioma cells. Further in vivo blood and brain PK study along with fluorescent microscopic examination of brain tissue will be carried out in experimental mice to estimate the potentiality of the optimized formulation both qualitatively and quantitatively to deliver LS successfully into brain tissue.