Dual kinase inhibitor for EGFR mutants and ErbB2 limit breast cancer.

Mutations in the epidermal growth factor receptor (EGFR) have been found in more than 10% of non-small cell lung cancer (NSCLC) patients in North America. The vast majority of these differences are L858R point mutations in Exon 21. Currently, monoclonal antibodies directed against the extracellular domain of EGFR or small molecule/tyrosine kinase inhibitors (TKI) are the stalwarts of NSCLC therapy. Resistance, however, gradually develops because of the T790 mutation towards first and second generation TKIs. The third generation TKI AZD9291 (Osimertinib) has a high affinity for both activating and the acquired resistant mutation (T790 M) in EGFR, with a low affinity towards wild-type EGFR. Recent research, however, suggests that the EGFR (C797S) mutation in the tyrosine kinase domain is a likely cause of resistance to AZD9291. Another significant transformation mechanism associated with this resistance is erbB2 amplification. Our laboratory has developed a small kinase inhibitor, ER121 (MW: ∼500), that inhibits the erbB2/HER2 tyrosine kinases in addition to the EGFR C797S mutations. We have identified a TKI, ER121 targeting the mutant EGFR(T790 M). Using in vitro and in vivo models, examined the efficacy of ER121 on mutant EGFR cell lines. This has enabled us to establish that ER121 is well tolerated when administered orally and produces significant inhibitory activity against human cancers generated by mutant EGFR and amplified ErbB2.

HED, a Human-Engineered Domain, Confers a Unique Fc-Binding Activity to Produce a New Class of Humanized Antibody-like Molecules.

Our laboratory has identified and developed a unique human-engineered domain (HED) structure that was obtained from the human Alpha-2-macroglobulin receptor-associated protein based on the three-dimensional structure of the Z-domain derived from Staphylococcal protein A. This HED retains µM binding activity to the human IgG1CH2-CH3 elbow region. We determined the crystal structure of HED in association with IgG1's Fc. This demonstrated that HED preserves the same three-bundle helix structure and Fc-interacting residues as the Z domain. HED was fused to the single chain variable fragment (scFv) of mAb 4D5 to produce an antibody-like protein capable of interacting with the p185Her2/neu ectodomain and the Fc of IgG. When further fused with murine IFN-γ (mIFN-γ) at the carboxy terminus, the novel species exhibited antitumor efficacy in vivo in a mouse model of human breast cancer. The HED is a novel platform for the therapeutic utilization of engineered proteins to alleviate human disease.

The circadian clock protects against ionizing radiation-induced cardiotoxicity.

Radiation therapy (RT) is commonly used to treat solid tumors of the breast, lung, and esophagus; however, the heart is an unintentional target of ionizing radiation (IR). IR exposure to the heart results in chronic toxicities including heart failure. We hypothesize that the circadian system plays regulatory roles in minimizing the IR-induced cardiotoxicity. We treated mice in control (Day Shift), environmentally disrupted (Rotating Shift), and genetically disrupted (Per 1/2 mutant) circadian conditions with 18 Gy of IR to the heart. Compared to control mice, circadian clock disruption significantly exacerbated post-IR systolic dysfunction (by ultrasound echocardiography) and increased fibrosis in mice. At the cellular level, Bmal1 protein bound to Atm, Brca1, and Brca2 promoter regions and its expression level was inversely correlated with the DNA damage levels based on the state of the clock. Further studies with circadian synchronized cardiomyocytes revealed that Bmal1 depletion increased the IR-induced DNA damage and apoptosis. Collectively, these findings suggest that the circadian clock protects from IR-induced toxicity and potentially impacts RT treatment outcome in cancer patients through IR-induced DNA damage responses.

PRMT5 and Tip60 Modify FOXP3 Function in Tumor Immunity.

Posttranslational modifications (PTMs) such as protein arginine methylation are involved in the regulation of diverse cellular processes such as epigenetic modifications, DNA damage response (DDR), RNA processing, signal transduction, and immune responses. Protein methyltransferases (PRMTs), which mediate arginine methylation, have been studied because of their dysregulation in several diseases. PRMT5, a type II arginine methyltransferase is relevant to cancer progression. Inhibition/deletion of PRMT5 augments tumor immunity by modulating Tip60 histone acetyltransferase activity and FOXP3 levels and limits the inhibitory function of T regulatory (Treg) cells, providing an approach to treat human cancers in an effective and exclusive manner. The activity of PRMT5 is regulated at various levels involving interaction with regulatory proteins, PTM modifications and noncoding RNA. Several PRMT5 inhibitors have been developed and are undergoing clinical trials or are in the preclinical phases. The current review concerns the regulation, biological functions, and therapeutic approaches for targeting PRMT5 with a focus on its role in tumor immunity. Critically, PRMT5 regulates the expression of Tip60 which we have shown is needed for FOXP3 regulatory interactions with DNA.

Notch signaling inhibition protects against LPS mediated osteolysis.

Chronic inflammatory responses have profound effects on the differentiation and activity of both the bone-forming osteoblasts and bone-resorbing osteoclasts. Importantly, inflammatory bone diseases characterized by clinical osteolysis promote bone resorption and decrease bone formation by uncoupling the process in favor of excess resorption. Notch signaling regulates osteoclast development and thus its manipulation has the potential to suppress resorptive potential. Here, we have utilized a genetic model of Notch inhibition in osteoclasts by expression of dnMAML to prevent formation of transcriptional complex essential for downstream Notch signaling. Using this model and LPS as a tool for experimental inflammatory osteolysis, we have demonstrated that dnMAML-expressing osteoclasts exhibited significantly lower maturation and resorption/functional potential ex vivo using TRAP staining and calcium phosphate coated surfaces. Moreover, we observed that while LPS stimulated the formation of wildtype osteoclasts pre-treated with RANKL, dnMAML expression produced resistance to osteoclast maturation after LPS stimulation. Genetically, Notch-inhibited animals showed a significantly lower TRAP and CTX-1 levels in serum after LPS treatment compared to the control groups in addition to a marked reduction in osteoclast surfaces in calvaria sections. This report provides evidence for modulation of Notch signaling activity to protect against inflammatory osteolysis. Taken together, the findings of this study will help guide the development of Notch signaling-based therapeutic approaches to prevent bone loss.

Docetaxel in cationic lipid nanocapsules for enhanced in vivo activity.

The usefulness of Docetaxel (DT) as an anti-cancer agent is limited to parenteral route owing to its very poor oral bioavailability. Thus, to improve its oral efficacy, DT was loaded in novel cationic lipid nanocapsules (DT CLNC). The DT CLNC possessed size of 130-150 nm, zeta potential of +72mV, adequate DT loading and over 95% encapsulation efficiency. TEM revealed capsular structure of DT CLNC. Lipolysis study indicated improved solubilization of DT by nanocapsules in comparison to DT solution. DT CLNC exhibited significantly higher release of DT in comparison to DT solution during in vitro permeation studies employing non-reverted rat-intestinal sac. Superior uptake of DT in zebra fishes exposed to DT CLNC resulted in greater apoptosis-based cell death as compared to those exposed to DT solution. This correlated well with the significantly superior (p < 0.05) anti-angiogenic activity of DT CLNC system over DT solution, in zebra fish model. DT CLNC also inhibited tumor growth in melanoma cell line induced tumors in C57BL/6 mice significantly, as compared to DT solution (p < 0.05). The DT CLNC system demonstrated adequate stability, with tremendous potential to improve oral efficacy of DT and can serve as an alternative to existing DT formulations available commercially for parenteral use.

Unraveling the cytotoxic potential of Temozolomide loaded into PLGA nanoparticles.

BACKGROUND: Nanotechnology has received great attention since a decade for the treatment of different varieties of cancer. However, there is a limited data available on the cytotoxic potential of Temozolomide (TMZ) formulations. In the current research work, an attempt has been made to understand the anti-metastatic effect of the drug after loading into PLGA nanoparticles against C6 glioma cells.Nanoparticles were prepared using solvent diffusion method and were characterized for size and morphology. Diffusion of the drug from the nanoparticles was studied by dialysis method. The designed nanoparticles were also assessed for cellular uptake using confocal microscopy and flow cytometry. RESULTS: PLGA nanoparticles caused a sustained release of the drug and showed a higher cellular uptake. The drug formulations also affected the cellular proliferation and motility. CONCLUSION: PLGA coated nanoparticles prolong the activity of the loaded drug while retaining the anti-metastatic activity.

Is an alternative drug delivery system needed for docetaxel? The role of controlling epimerization in formulations and beyond.

PURPOSE: The presence of 7-epidocetaxel in docetaxel injection and in vivo epimerisation has been reported to be the cause for development of tumor resistance to chemotherapy including docetaxel by inducing tumor cell protein cytochrome P450 1B1. The objective of this study was to determine systemic toxicity of Taxotere® containing 10% 7-epidocetaxel and to develop PEGylated liposomal injection that could resist epimerization in vivo. Another need for PEGylated liposomal delivery of docetaxel is to avoid reported hypersensitivity reactions of marketed products like Taxotere® and Duopafei® containing high concentration of tween-80. METHODS: The PEGylated liposomes loaded with docetaxel were prepared using thin film hydration method. The in vivo toxicity of Taxotere® containing 10% 7-epimer was studied in B16F10 experimental metastasis model. RESULTS: B16F10 experimental metastasis model using C57BL/6 mice injected with Taxotere® containing 10% 7-epimer showed higher weight loss as compared to Taxotere® containing no epimer at single dose of 40 mg/kg indicating higher systemic toxicity. Incubation of PEGylated liposomes with phosphate buffer saline (pH 7.4) containing 0.1% w/v Tween-80 for 48 h showed better resistance to docetaxel degradation when compared with Taxotere® injection indicating better in vivo stability of liposomal docetaxel. In addition, PEGylated liposomes showed enhanced in vitro cytotoxicity, against A549 and B16F10 cells, than Taxotere®. CONCLUSION: We can therefore expect less in vivo conversion of liposomal loaded docetaxel into 7-epimer, more passive targeting to tumor tissues, decreased 7-epimer induced systemic toxicity and tumor resistance to chemotherapy compared to Taxotere®. Further in vivo studies are needed to ascertain these facts.

Unravelling the antimetastatic potential of pentoxifylline, a methylxanthine derivative in human MDA-MB-231 breast cancer cells.

Pentoxifylline (PTX), a methylxanthine derivative is a non-steroidal immunomodulating agent with unique hemorheologic properties. It is used in the treatment of intermittent claudication as it increases the amount of oxygen reaching tissues by increasing the flexibility of red blood cells. Recently, it has also shown to exhibit anti-metastatic and anti-angiogenic activities in B16F10 melanoma cells both in vitro as well as in vivo. As per the reports, the choice of drug in the treatment of breast cancer is paclitaxel, but the major limitation is its toxicity. However, the effects of PTX on metastatic processes in breast cancer are not currently known. Therefore, in this study, we have examined the effect of PTX in MDA-MB-231 human breast cancer cells. The MTT assay showed dose- and time-dependent decreases in cellular proliferation. The non-toxic concentration of PTX selected were 1, 2.5 and 5 mM for 24 h. PTX induced a G0-G1 cell-cycle arrest leading to apoptosis. Further, it affected adhesion to both the matrigel and collagen type-IV in a time- and dose-dependent manner. The PTX impeded the migration of MDA-MB-231 cells and also decreased the activities of both MMP-2 and MMP-9. Thus, PTX at non-toxic doses affected cellular proliferation, adhesion, migration and invasion. These results demonstrate its anti-metastatic effect on MDA-MB-231 cells, and further studies need to be carried out to understand the mechanism of action.

Regulatory T Cells: Regulation of Identity and Function.

T regulatory cells suppress a variety of immune responses to self-antigens and play a role in peripheral tolerance maintenance by limiting autoimmune disorders, and other pathological immune responses such as limiting immune reactivity to oncoprotein encoded antigens. Forkhead box P3 (FOXP3) expression is required for Treg stability and affects functional activity. Mutations in the master regulator FOXP3 and related components have been linked to autoimmune diseases in humans, such as IPEX, and a scurfy-like phenotype in mice. Several lines of evidence indicate that Treg use a variety of immunosuppressive mechanisms to limit an immune response by targeting effector cells, including secretion of immunoregulatory cytokines, granzyme/perforin-mediated cell cytolysis, metabolic perturbation, directing the maturation and function of antigen-presenting cells (APC) and secretion of extracellular vesicles for the development of immunological tolerance. In this review, several regulatory mechanisms have been highlighted and discussed.

FOXP3 and Tip60 Structural Interactions Relevant to IPEX Development Lead to Potential Therapeutics to Increase FOXP3 Dependent Suppressor T Cell Functions.

Regulatory T (Treg) cells play a role in the maintenance of immune homeostasis and are critical mediators of immune tolerance. The Forkhead box P3 (FOXP3) protein acts as a regulator for Treg development and function. Mutations in the FOXP3 gene can lead to autoimmune diseases such as Immunodysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX) syndrome in humans, often resulting in death within the first 2 years of life and a scurfy like phenotype in Foxp3 mutant mice. We discuss biochemical features of the FOXP3 ensemble including its regulation at various levels (epigenetic, transcriptional, and post-translational modifications) and molecular functions. The studies also highlight the interactions of FOXP3 and Tat-interacting protein 60 (Tip60), a principal histone acetylase enzyme that acetylates FOXP3 and functions as an essential subunit of the FOXP3 repression ensemble complex. Lastly, we have emphasized the role of allosteric modifiers that help stabilize FOXP3:Tip60 interactions and discuss targeting this interaction for the therapeutic manipulation of Treg activity.

Challenges in Detection of Serum Oncoprotein: Relevance to Breast Cancer Diagnostics.

Breast cancer is a highly prevalent malignancy that shows improved outcomes with earlier diagnosis. Current screening and monitoring methods have improved survival rates, but the limitations of these approaches have led to the investigation of biomarker evaluation to improve early diagnosis and treatment monitoring. The enzyme-linked immunosorbent assay (ELISA) is a specific and robust technique ideally suited for the quantification of protein biomarkers from blood or its constituents. The continued clinical relevancy of this assay format will require overcoming specific technical challenges, including the ultra-sensitive detection of trace biomarkers and the circumventing of potential assay interference due to the expanding use of monoclonal antibody (mAb) therapeutics. Approaches to increasing the sensitivity of ELISA have been numerous and include employing more sensitive substrates, combining ELISA with the polymerase chain reaction (PCR), and incorporating nanoparticles as shuttles for detection antibodies and enzymes. These modifications have resulted in substantial boosts in the ability to detect extremely low levels of protein biomarkers, with some systems reliably detecting antigen at sub-femtomolar concentrations. Extensive utilization of mAb therapies in oncology has presented an additional contemporary challenge for ELISA, particularly when both therapeutic and assay antibodies target the same protein antigen. Resolution of issues such as epitope overlap and steric hindrance requires a rational approach to the design of diagnostic antibodies that takes advantage of modern antibody generation pipelines, epitope binning techniques and computational methods to strategically target biomarker epitopes. This review discusses technical strategies in ELISA implemented to date and their feasibility to address current constraints on sensitivity and problems with interference in the clinical setting. The impact of these recent advancements will depend upon their transformation from research laboratory protocols into facile, reliable detection systems that can ideally be replicated in point-of-care devices to maximize utilization and transform both the diagnostic and therapeutic monitoring landscape.

Suppression of Notch Signaling in Osteoclasts Improves Bone Regeneration and Healing.

Owing to the central role of osteoclasts in bone physiology and remodeling, manipulation of their maturation process provides a potential therapeutic strategy for treating bone diseases. To investigate this, we genetically inhibited the Notch signaling pathway in the myeloid lineage, which includes osteoclast precursors, using a dominant negative form of MAML (dnMAML) that inhibits the transcriptional complex required for downstream Notch signaling. Osteoclasts derived from dnMAML mice showed no significant differences in early osteoclastic gene expression compared to the wild type. Further, these demonstrated significantly lowered resorption activity using bone surfaces while retaining their osteoblast stimulating ability using ex vivo techniques. Using in vivo approaches, we detected significantly higher bone formation rates and osteoblast gene expression in dnMAML cohorts. Further, these mice exhibited increased bone/tissue mineral density compared to wild type and larger bony calluses in later stages of fracture healing. These observations suggest that therapeutic suppression of osteoclast Notch signaling could reduce, but not eliminate, osteoclastic resorption without suppression of restorative bone remodeling and, therefore, presents a balanced paradigm for increasing bone formation, regeneration, and healing. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2089-2103, 2019.

Thermosensitive PLA based nanodispersion for targeting brain tumor via intranasal route.

Delivery of drugs to the brain via nasal route has been studied by many researchers. However, low residence time, mucociliary clearance and enzymatically active environment of nasal cavity pose many challenges to successful nasal delivery of drugs. We aim to deliver methotrexate by designing thermosensitive nanodispersion exhibiting enhanced residence time in nasal cavity and bypassing the blood brain barrier (BBB). PLA nanoparticles were developed using solvent evaporation technique. The developed nanoparticles were further dispersed in prepared thermosensitive vehicle of poloxamer 188 and Carbopol 934 to impart the property of increased residence time. The formulated nanoparticles demonstrated no interaction with the simulated nasal fluids (SNF), mucin, serum proteins and erythrocytes which demonstrate the safety of developed formulation for nasal administration. The penetration property of nanoparticles though the nasal mucosa was higher than the pure drug due to low mucociliary clearance. The developed nanoparticles diffused though the membrane pores and rapidly distributed into the brain portions compared to the pure drug. There was detectable and quantifiable amount of drug seen in the brain as demonstrated by in vivo brain distribution studies with considerably low amount of drug deposition in the lungs. The pharmacokinetic parameters demonstrated the enhancement in circulation half life, area under curve (AUC) and Cmax of the drug when administered intranasal in encapsulated form. Thus, the thermosensitive nanodispersions are surely promising delivery systems for delivering anticancer agents though the nasal route for potential treatment of brain tumors.

Surface-coated PLA nanoparticles loaded with temozolomide for improved brain deposition and potential treatment of gliomas: development, characterization and in vivo studies.

Hydrophobicity of PLA nanoparticles makes them a good substrate for macrophageal and reticulo-endothelial system uptake. Long-circulating properties can be imparted to these particles by coating them with hydrophilic stabilizers. Surface-modified PLA nanoparticles loaded with anti-cancer agent temozolomide were fabricated by solvent evaporation method and coated with surface modifiers. Selection of the surface modifier was based upon uptake of nanoparticles by K9 cells (liver cells). The particles were prepared and characterized for various physicochemical properties using transmission electron microscopy, differential scanning calorimetry, powder X-ray diffraction and in vitro dissolution studies. In vitro BBB permeation studies were performed using the co-culture model developed by using Madin-Darby canine kidney and C6 glioma cells as endothelial and glial cells, respectively. In vitro C6 glioma cell cytotoxicity, cellular proliferation, cellular migration and cellular uptake studies due to developed nanoparticles was assessed. In vivo studies such as pharmacokinetics, qualitative and quantitative biodistribution studies were performed for the developed nanoparticles. Drug-loaded nanoparticles with entrapment efficiency of 50% were developed. PEG-1000 and polysorbate-80 coated nanoparticles were least taken up by the liver cells. Characterization of the nanoparticles revealed formation of spherical shape nanoparticles, with no drug and excipient interaction. In vivo pharmacokinetics of developed nanoparticles depicted enhancement of half-life, area under the curve and mean residence time of the drug. Qualitative and quantitative biodistribution studies confirmed enhanced permeation of the drug into the brain upon loading into nanoparticles with less deposition in the highly perfused organs like lung, liver, spleen, heart and kidney.

Delineating the anti-metastatic potential of pentoxifylline in combination with liposomal doxorubicin against breast cancer cells.

Breast cancer remains the second most prevalent cancer worldwide. Several anticancer drugs are being currently used in the treatment of breast cancer. However, owing to high cytotoxicity, induced resistance and cost ineffectiveness, there is an urgent need to develop newer therapeutic regimens that limit the current problems. One of the approaches in this regard is the formulation of combination therapies whereby multiple drugs are being delivered at relatively lesser dose that surely confines the aforesaid problems. In this purview, we had evaluated the effects of pentoxifylline, a methylxanthine derivative and liposomal doxorubicin (Lipodox), an anthracycline in combination to evaluate their anti-metastatic activities both in vitro and in vivo against breast cancer cells. The combination regime exhibited synergistic activity and inhibited cellular proliferation to a greater extent with regard to each drug used alone.

Pentoxifylline regulates the cellular adhesion and its allied receptors to extracellular matrix components in breast cancer cells.

Pentoxifylline (PTX) is a methylxanthine derivative that improves blood flow by decreasing its viscosity. Being an inhibitor of platelet aggregation, it can thus reduce the adhesiveness of cancer cells prolonging their circulation time. This delay in forming secondary tumours makes them more prone to immunological surveillance. Recently, we have evaluated its anti-metastatic efficacy against breast cancer, using MDA-MB-231 model system. In view of this, we had ascertained the effect of PTX on adhesion of MDA-MB-231 cells to extracellular matrix components (ECM) and its allied receptors such as the integrins. PTX affected adhesion of breast cancer cells to matrigel, collagen type IV, fibronectin and laminin in a dose dependent manner. Further, PTX showed a differential effect on integrin expression profile. The experimental metastasis model using NOD-SCID mice showed lesser tumour island formation when treated with PTX compared to the control. These findings further substantiate the anti-adhesive potential of PTX in breast cancer and warrant further insights into the functional regulation.

Anti-neuropilin 1 antibody Fab' fragment conjugated liposomal docetaxel for active targeting of tumours.

Neuropilin-1, a transmembrane receptor entailed in wide range of human tumour cell lines and diverse neoplasms, mediates the effects of VEGF and Semaphorins during the processes of cellular proliferation, survival and migration. In view of this, we had developed and evaluated in vitro and in vivo efficacy of anti-neuropilin-1 immunoliposomes against neuropilin-1 receptor expressing tumours. The PEGylated liposomes loaded with docetaxel were prepared using thin film hydration method. Functionalised PEGylated liposomes were prepared using post-insertion technique. Anti-neuropilin-1 immunoliposomes were prepared by covalently conjugating Fab' fragments of neuropilin-1 antibody to functionalised PEGylated liposomes via thioether linkage. In vivo evaluation of Taxotere and liposomal formulations was performed using intradermal tumour model to demonstrate anti-angiogenic and tumour regression ability. The modified Fab' fragments and immunoliposomes were found to be immunoreactive against A549 cells. Further, docetaxel loaded PEGylated liposomes and PEGylated immunoliposomes demonstrated higher in vitro cytotoxicity than Taxotere formulation at the same drug concentration and exposure time. The live imaging showed distinctive cellular uptake of functional immunoliposomes. Further, significant decrease in micro-blood vessel density and tumour volumes was observed using bio-engineered liposomes. The results clearly highlight the need to seek neuropilin-1 as one of the prime targets in developing an anti-angiogenic therapy.

Tamoxifen guided liposomes for targeting encapsulated anticancer agent to estrogen receptor positive breast cancer cells: in vitro and in vivo evaluation.

Tamoxifen (TMX), an estrogen receptor (ER) antagonist, incorporated at surface of liposomes loaded with Doxorubicin (DOX), was hypothesized to serve as ligand for targeting overexpressed ERs on surface and cytosol of breast cancer cells, in addition to its synergism with DOX in killing MCF-7 cells. The TMX-DOX liposomes demonstrated mean size of 188.8±2.2nm and positive potential of+47mV, both suitable for better cellular interaction. TMX-DOX liposomes sustained DOX release in vitro (25.9%) in pH 7.4 at 48h, in comparison with 64.5% DOX release at pH 5.5. In vitro cell line studies demonstrated that TMX-DOX liposomes were more cytotoxic to ER+ve MCF-7 cells as compared to DOX liposomes, DOX solution and TMX-DOX solution (P<0.05). However, there was no statistical difference in cyto-toxicity of TMX-DOX liposomes and DOX liposomes towards ER-ve MDA-MB-231 cells. Flow cytometry and confocal studies in MCF-7 cells revealed greater cell and nuclear uptake of DOX, with TMX guided liposomes as compared to DOX liposomes and DOX solution. TMX-DOX liposomes demonstrated significantly increased inhibition of MCF-7 cell based tumor growth in nude mice (P<0.05) in comparison to DOX solution and DOX liposomes, indicative of target specificity and higher DOX accumulation at tumor site.

Liposomes for targeting hepatocellular carcinoma: use of conjugated arabinogalactan as targeting ligand.

Present study investigates the potential of chemically modified (Shah et al., 2013) palmitoylated arabinogalactan (PAG) in guiding liposomal delivery system and targeting asialoglycoprotein receptors (ASGPR) which are expressed in hepatocellular carcinoma (HCC). PAG was incorporated in liposomes during preparation and doxorubicin hydrochloride was actively loaded in preformed liposomes with and without PAG. The liposomal systems with or without PAG were evaluated for in vitro release, in vitro cytotoxicity, in vitro cell uptake on ASGPR(+) cells, in vivo pharmacokinetic study, in vivo biodistribution study, and in vivo efficacy study in immunocompromised mice. The particle size for all the liposomal systems was below 200 nm with a negative zeta potential. Doxorubicin loaded PAG liposomes released significantly higher amount of doxorubicin at pH 5.5 as compared to pH 7.4, providing advantage for targeted tumor therapy. Doxorubicin in PAG liposomes showed superior cytotoxicity on ASGPR(+) HepG2 cells as compared to ASGPR(-), MCF7, A549, and HT29 cells. Superior uptake of doxorubicin loaded PAG liposomes as compared to doxorubicin loaded conventional liposomes was evident in confocal microscopy studies. Higher AUC in pharmacokinetic study and higher deposition in liver was observed for PAG liposomes compared to conventional liposomes. Significantly higher tumor suppression was noted in immunocompromised mice for mice treated with PAG liposomes as compared to the conventional liposomes. Targeting ability and superior activity of PAG liposomes is established pre-clinically suggesting potential of targeted delivery system for improved treatment of HCC.