Triple negative breast cancer and non-small cell lung cancer: Clinical challenges and nano-formulation approaches.

Triple negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC) are amongst the most aggressive forms of solid tumors. TNBC is highlighted by absence of genetic components of progesterone receptor, HER2/neu and estrogen receptor in breast cancer. NSCLC is characterized by integration of malignant carcinoma into respiratory system. Both cancers are associated with poor median and overall survival rates with low progression free survival with high incidences of relapse. These cancers are characterized by tumor heterogeneity, genetic mutations, generation of cancer-stem cells, immune-resistance and chemoresistance. Further, these neoplasms have been reported for tumor cross-talk into second primary cancers for each other. Current chemotherapeutic regimens include usage of multiple agents in tandem to affect tumor cells through multiple mechanisms with various such combinations being clinically tested. However, lack of controlled delivery and effective temporospatial presence of chemotherapeutics has resulted in suboptimal therapeutic response. Consequently, passive targeted albumin bound paclitaxel and PEGylated liposomal doxorubicin have been clinically used and tested with newer drugs for improved therapeutic efficacy in these cancers. Active targeting of nanocarriers against surface overexpressed proteins in both neoplasms have been explored. However, use of single agent nanoparticulate formulations against both cancers have failed to elicit desired outcomes. This review aims to identify clinical unmet need in these cancers while establishing a correlation with tested nano-formulation approaches and issues with preclinical to clinical translation. Lipid and polymer-based drug-drug and drug-gene combinatorial nanocarriers delivering multiple chemotherapeutics simultaneously to desired site of action have been detailed. Finally, emerging opportunities such as pharmacological targets (immune check point and epigentic modulators) as well as gene-based modulation (siRNA/CRISPR/Cas9) and the nano-formulation challenges for effective treatment of both cancers have been explored.

Synergistic co-loading of vincristine improved chemotherapeutic potential of pegylated liposomal doxorubicin against triple negative breast cancer and non-small cell lung cancer.

The current work aims to explore the biological characteristics of vincristine synergistic co-loading into pegylated liposomal doxorubicin in non-indicated modalities of non-small cell lung cancer (NSCLC) and triple negative breast cancer (TNBC). The combinatorial liposome prepared by active co-loading of the drugs against modified ammonium ion gradient exhibited 95% encapsulation of both drugs. The cellular uptake studies using confocal microscopy and flow cytometry showed significantly increased uptake of dual drug formulation as against liposomal doxorubicin. The co-loaded liposome formulation had significantly increased cell cycle arrest in G2/M phase with subsequent apoptosis and reduced cell viability in both tumor cell lines than doxorubicin liposome. This carrier exhibited similar acute toxicity, pharmacokinetic and tissue distribution profiles with significant increase in tumor regression as compared to liposomal doxorubicin. These results indicate that co-encapsulation of vincristine into clinically used pegylated liposomal doxorubicin significantly improved in-vitro and in-vivo therapeutic efficacy against NSCLC and TNBC.

Optimization and efficacy study of synergistic vincristine coloaded liposomal doxorubicin against breast and lung cancer.

Aim: To improve the efficacy of poly-ethylene glycol (PEG)ylated liposomes coloaded with doxorubicin and vincristine against triple-negative breast cancer (TNBC) and non-small-cell lung cancer (NSCLC). Methods: The combinatorial index of the drugs was established using the Chou-Talalay method in MDA-MB-231 and A549 cell lines. The most effective ratio was co-encapsulated in factorial design optimized nanoliposomes which were characterized for similarity to clinical standard and evaluated in vitro and in vivo for therapeutic efficacy. Results & conclusion: The formulation exhibited more than 95% co-encapsulation, a size of 95.74 ± 2.65 nm and zeta potential of -9.17 ± 1.19 mV while having no significant differences in physicochemical and biochemical characteristics as compared with the clinical standard. Efficacy evaluation studies showed significantly improved cytotoxicity and tumor regression compared with liposomal doxorubicin indicating improvement in efficacy against TNBC and NSCLC.

Dual drug loaded liposome bearing apigenin and 5-Fluorouracil for synergistic therapeutic efficacy in colorectal cancer.

Multidrug-based combinatorial therapeutic regiments which target multiple pathways simultaneously are being utilized as a therapeutic strategy of choice due to reduction in toxicity profile and enhancement of therapeutic index of the individual drugs. 5-Fluorouracil is a clinically approved drug which has limited response rate in the realm of colorectal cancer amelioration, hence our study aims to improve its efficacy by aiming the simultaneous delivery of 5-Flurouracil and apigenin which is naturally occurring flavone abundantly present in fruit and vegetables through a single liposome to combat and control colorectal cancer effectively in-vitro and in-vivo. The liposomal nanocarrier bearing the anti-tumorigenic agent apigenin was designed in this study in order to improve the bioavailability of the flavone while at the same time achieve combinatorial drug regime with 5- Fluorouracil. This study reports the synthesis and production of a relatively robust dual drug-loaded liposomal formulation by modified thin film hydration method which substantially entraps both the drugs. Even though there have been reports of the combinatorial regimen involving apigenin and 5-Flurouracil our study reports the optimal molar ratio for effective synergistic therapeutic application of this drug combination to alleviate colorectal cancer. The cytotoxicity and cellular effects of individual, combinatorial free drugs and their liposomal counterparts tested against two human colon cancer cell lines revealed significantly higher cytotoxicity of the dual-drug liposomes. The dual-drug liposomes demonstrated enhanced inhibition of angiogenesis, better reduction in cell proliferation and increased apoptotic potential. Cell signaling studies indicating a significant upregulation of pAMPK and activity against downstream targets by dual drug liposomes suggested its role in the reversal of Warburg effect. The formulation was tested in a preclinical setting in nude mice tumor xenograft model and was found to have greater anti-neoplastic and anti-tumorigenic effect. The study indicated that the increased chemotherapeutic potential in vivo was due to the passive targeting achieved by the liposomal drug loaded nano-carrier and the synergistic effect of apigenin in 5-Fluorouracil treatment offers a new attractive alternative to enhance the therapeutic potency of drugs and paves way for potential clinical applications.

Combinatorial nanocarriers against drug resistance in hematological cancers: Opportunities and emerging strategies.

Hematological cancers are a group of malignancies affecting human hematopoietic and lymphoid tissues. Although the patients respond to treatment regimen during initial phases, the hematoma tumor heterogeneity results in the presence of some minimal disease residue thereby exhibiting remission, relapses or refractoriness in disease conditions leading to poor overall survival period. The current therapeutic standard practices involve blending of conventional agents with novel targeting agents or immune-therapeutics in a cocktail to effectively reap the benefits of drugs acting through multiple signaling pathways. Considerable evaluation of the risk benefit ratio on part of clinicians is necessitated to select the best optimum therapy considering the high incidences of drug resistance. This drug resistance may be attributed to faulty upregulation or mutation of multiple drug resistance regulating genes, increased tumor cell immune system cross talk, increased expression of drug efflux pump inducers and inhibition of apoptosis among others. Conventional single drug nanotherapeutics as modulators of drug resistance have already clinically exhibited their potential by passively delivering the active cargo to desired targets in hematological neoplasms. However, with the ever-growing clinical failures of such therapies, the landscape of hematological cancer treatment has seen a plethora of changes in the last few years. The two towering changes in the treatment has been the approval of combinatorial drug nanocarrier Vyxeos™ and chimeric antigen receptor T cell (CAR-T) therapy Kymriah™ as well as Yescarta™. The approval of CAR-T therapy not only resulted in a paradigm shift in the avenues of blood cancer treatment towards personalized approaches but also saddled it with questions of economic viability and effectiveness in the entire spectrum of such neoplasms. Under such conditions, combinatorial drug nanocarriers encompassing synergistic ratios of clinically effective drug combinations affording temporal and spatial control present an exciting approach to overcome these drug resistance modalities. This platform provides increased chances of therapeutic in-vitro in-vivo correlation along with minimization of drug resistance and associated disease relapse conditions. The present review intends to present the current preclinical and clinical advances in combinatorial nanocarrier mediated management of drug resistance in hematological cancers.

Nanoliposomal delivery of cytosolic phospholipase A2 inhibitor arachidonyl trimethyl ketone for melanoma treatment.

Drug resistance and toxicity are major limitations of cancer treatment and frequently occurs during melanoma therapy. Nanotechnology can decrease drug resistance by improving drug delivery, with limited toxicity. This study details the development of nanoparticles containing arachidonyl trifluoromethyl ketone (ATK), a cytosolic phospholipase A2 inhibitor, which can inhibit multiple key pathways responsible for the development of recurrent resistant disease. Free ATK is toxic, limiting its efficacy as a therapeutic agent. Hence, a novel nanoliposomal delivery system called NanoATK was developed, which loads 61.7% of the compound and was stable at 4oC for 12 weeks. The formulation decreased toxicity-enabling administration of higher doses, which was more effective at inhibiting melanoma cell growth compared to free-ATK. Mechanistically, NanoATK decreased cellular proliferation and triggered apoptosis to inhibit melanoma xenograft tumor growth without affecting animal weight. Functionally, it inhibited the cPLA2, AKT, and STAT3 pathways. Our results suggest the successful preclinical development of a unique nanoliposomal formulation containing ATK for the treatment of melanoma.

Enhanced chemotherapeutic efficacy of apigenin liposomes in colorectal cancer based on flavone-membrane interactions.

Recent endeavors in exploiting vast array of natural phytochemicals to ameliorate colorectal cancer led us to investigate apigenin, a naturally occurring dietary flavone as a potential chemo-therapeutic agent. The present study focuses on establishing apigenin as a potential chemotherapeutic agent for alleviating colorectal cancer and reports the development of a stable liposomal nanocarrier with high encapsulation of the hydrophobic flavone apigenin for enhanced chemotherapeutic effects. The enhanced pharmacological activity of apigenin has been assigned to its ability to interact and subsequently influence membrane properties which also resulted in optimal yield of a stable, rigidified, non-leaky nano-carrier with ideal release kinetics. Extensive testing of drug and its liposomal counterpart for potential clinical chemotherapeutic applications yielded hemocompatibility and cytocompatibility with normal fibroblast cells while enhanced antineoplastic activity was observed in tumor xenograft model. The increased chemotherapeutic potential of liposomal apigenin highlights the clinical potential of apigenin-based vesicles.

Probing the potential of apigenin liposomes in enhancing bacterial membrane perturbation and integrity loss.

Along with discovery of new antibacterial agents, it is important to develop novel drug delivery systems to effectively deliver drugs within bacterial cells for enhanced therapeutic activity. Liposomes have been extensively investigated as pharmaceutical carriers for improvement of therapeutic index of antimicrobial agents. The aim of this present study was to evaluate the antibacterial activity of free and liposomal formulation of apigenin, a plant based isoflavone and elucidate the mode of action. Distearoylphosphatidylcholine liposomes were prepared having nano-range particle size (104.3±1.8 nm), narrow particle distribution (0.204) and high encapsulation efficiency of apigenin (89.9±2.31%). Antibacterial activity of apigenin and efficacy of liposome-mediated apigenin delivery were determined from minimum inhibitory concentration values. Interaction studies using electron microscopy revealed adherence and fusion of liposomal apigenin with the bacteria causing membrane perturbation through reactive oxygen species generation which was evaluated by epi-fluorescence microscopy and fluorescence activated cell sorting. The interaction of apigenin liposomes with bacterial membrane increased intracellular drug concentration and thus, can be employed to deliver apigenin within cells to augment its antibacterial activity. Increased efficacy and hemocompatibility of this formulation paves way for future evaluation of underlying molecular mechanisms and in vivo testing for enhanced therapeutic effects.

Use of Nanotechnology to Develop Multi-Drug Inhibitors For Cancer Therapy.

Therapeutic agents that inhibit a single target often cannot combat a multifactorial disease such as cancer. Thus, multi-target inhibitors (MTIs) are needed to circumvent complications such as the development of resistance. There are two predominant types of MTIs, (a) single drug inhibitor (SDIs) that affect multiple pathways simultaneously, and (b) combinatorial agents or multi-drug inhibitors (MDIs) that inhibit multiple pathways. Single agent multi-target kinase inhibitors are amongst the most prominent class of compounds belonging to the former, whereas the latter includes many different classes of combinatorial agents that have been used to achieve synergistic efficacy against cancer. Safe delivery and accumulation at the tumor site is of paramount importance for MTIs because inhibition of multiple key signaling pathways has the potential to lead to systemic toxicity. For this reason, the development of drug delivery mechanisms using nanotechnology is preferable in order to ensure that the MDIs accumulate in the tumor vasculature, thereby increasing efficacy and minimizing off-target and systemic side effects. This review will discuss how nanotechnology can be used for the development of MTIs for cancer therapy and also it concludes with a discussion of the future of nanoparticle-based MTIs as well as the continuing obstacles being faced during the development of these unique agents.'

Poly(styrene-co-maleic acid)-based pH-sensitive liposomes mediate cytosolic delivery of drugs for enhanced cancer chemotherapy.

pH-responsive polymers render liposomes pH-sensitive and facilitate the intracellular release of encapsulated payload by fusing with endovascular membranes under mildly acidic conditions found inside cellular endosomes. The present study reports the use of high-molecular weight poly(styrene-co-maleic acid) (SMA), which exhibits conformational transition from a charged extended structure to an uncharged globule below its pK(1) value, to confer pH-sensitive property to liposomes. The changes in the co-polymer chain conformation resulted in destabilization of the liposomes at mildly acidic pH due to vesicle fusion and/or channel formation within the membrane bilayer, and ultimately led to the release of the encapsulated cargo. The vesicles preserved their pH-sensitivity and stability in serum unlike other polymer-based liposomes and exhibited no hemolytic activity at physiological pH. The lysis of RBCs at endosomal pH due to SMA-based liposome-induced alterations in the bilayer organization leading to spherocyte formation indicated the potential of these vesicles to mediate cytosolic delivery of bio-active molecules through endosome destabilization. The SMA-loaded liposomes exhibiting excellent cytocompatibility, efficiently delivered chemotherapeutic agent 5-Fluorouracil (5-FU) within colon cancer cells HT-29 in comparison to neat liposomes. This caused increased cellular-availability of the drug, which resulted in enhanced apoptosis and highlighted the clinical potential of SMA-based vesicles.

Probing molecular interactions of poly(styrene-co-maleic acid) with lipid matrix models to interpret the therapeutic potential of the co-polymer.

To understand and maximize the therapeutic potential of poly(styrene-co-maleic acid) (SMA), a synthetic, pharmacologically-active co-polymer, its effect on conformation, phase behavior and stability of lipid matrix models of cell membranes were investigated. The modes of interaction between SMA and lipid molecules were also studied. While, attenuated total reflection-Fourier-transform infrared (ATR-FTIR) and static (31)P nuclear magnetic resonance (NMR) experiments detected SMA-induced conformational changes in the headgroup region, differential scanning calorimetry (DSC) studies revealed thermotropic phase behavior changes of the membranes. (1)H NMR results indicated weak immobilization of SMA within the bilayers. Molecular interpretation of the results indicated the role of hydrogen-bond formation and hydrophobic forces between SMA and zwitterionic phospholipid bilayers. The extent of membrane fluidization and generation of isotropic phases were affected by the surface charge of the liposomes, and hence suggested the role of electrostatic interactions between SMA and charged lipid headgroups. SMA was thus found to directly affect the structural integrity of model membranes.

Effect of liposomal celecoxib on proliferation of colon cancer cell and inhibition of DMBA-induced tumor in rat model.

Celecoxib, a selective cyclooxygenase-2 inhibitor, has shown potential anticancerous activity against majority of solid tumors especially on patients with colon cancer. However, associations of serious side effects limit the usage of celecoxib in colon cancer treatment. To address this issue and provide an alternative strategy to increase the efficacy of celecoxib, liposomal formulation of celecoxib was prepared and characterized. Anticancer activity of liposomal celecoxib on colon cancer cell HCT 15 was evaluated in vitro. Furthermore, tumor inhibition efficiency by liposomal celecoxib was studied on 7,12-dimethyl benz(a)anthracene (DMBA)-induced tumor in rat model. In order to elucidate the antioxidant activity of celecoxib-loaded liposomes, antioxidant superoxide dismutase (SOD) generation and lipid peroxide (LPx) formation in both liver and kidney tissues were examined. Characterization of the formed unilamellar liposomes revealed the formation of homogeneous suspension of neutral (empty) or anionic (celecoxib-loaded) liposomes with a well-defined spherical shape which have a mean size of 103.5 nm (empty liposome) and 169 nm (liposomal celecoxib). High-performance liquid chromatography (HPLC) analysis and hemolytic assay demonstrated 46% of celecoxib entrapment efficiency and significantly low hemolysis, respectively. Liposomal celecoxib exhibited dose-dependent cytotoxicity and apoptotic activity against HCT 15 cells which are comparable to free celecoxib. In vivo study demonstrated inhibition of tumor growth. Biochemical analysis of the liposomal celecoxib-treated group significantly inhibited the LPx formation (oxygen-free radicals) and increased the activity of SOD. Our results present the potential of inhibiting colon cancer in vitro and DMBA-induced tumor in rat model in vivo by liposomal celecoxib.

RISUG: a potential candidate for the entry inhibitor group of antiretroviral drugs.

Entry inhibitors are a group of antiretroviral drug which prevents HIV from entering human immune cells. They include both fusion and attachment inhibitors. A hypothesis is put forward in which a new male contraceptive drug with proven antimicrobial property is proposed as a possible candidate for the entry inhibitor group of antiretroviral drugs. The proposed mechanism of action involves (i) interaction with gp120 and thereby preventing binding to CD4 and (ii) competitive binding with the viral glycoprotein and inhibit the glycoprotein - cell surface glyocosaminoglycan Heparan Sulfate (HS) interaction. A new drug RISUG (Reversible Inhibition of Sperm Under Guidance) presently undergoing Phase III clinical trials throughout India for its contraceptive effect in male has also antimicrobial actions. RISUG is a chemical complex of styrene maleic anhydride (SMA(AN)) and dimethyl sulfoxide. On injection into the vas deferens, it reacts with the components of intravas fluid, the spermatic fluid and gets converted to styrene maleic acid (SMA(AC)) and breakdown products like mandelic acid. An anti HIV activity of RISUG is likely due to its electrical charge and mandelic acid generation. For experimental validation HIV in vitro assays can be performed which will involve infectivity assays, luciferase assay and soluble gp120 assays. A positive result from the studies will validate the hypothesis.