Coumarin as an Elite Scaffold in Anti-Breast Cancer Drug Development: Design Strategies, Mechanistic Insights, and Structure-Activity Relationships.

Breast cancer is the most common cancer among women. Currently, it poses a significant threat to the healthcare system due to the emerging resistance and toxicity of available drug candidates in clinical practice, thus generating an urgent need for the development of new potent and safer anti-breast cancer drug candidates. Coumarin (chromone-2-one) is an elite ring system widely distributed among natural products and possesses a broad range of pharmacological properties. The unique distribution and pharmacological efficacy of coumarins attract natural product hunters, resulting in the identification of numerous natural coumarins from different natural sources in the last three decades, especially those with anti-breast cancer properties. Inspired by this, numerous synthetic derivatives based on coumarins have been developed by medicinal chemists all around the globe, showing promising anti-breast cancer efficacy. This review is primarily focused on the development of coumarin-inspired anti-breast cancer agents in the last three decades, especially highlighting design strategies, mechanistic insights, and their structure-activity relationship. Natural coumarins having anti-breast cancer efficacy are also briefly highlighted. This review will act as a guideline for researchers and medicinal chemists in designing optimum coumarin-based potent and safer anti-breast cancer agents.

Hepatic LRP-1 plays an important role in amyloidosis in Alzheimer's disease mice: Potential role in chronic heavy alcohol feeding.

BACKGROUND: Hepatic lipoprotein receptor-related protein 1 (LRP-1) plays a central role in peripheral amyloid beta (Aß) clearance, but its importance in Alzheimer's disease (AD) pathology is understudied. Our previous work showed that intragastric alcohol feeding to C57BL/6 J mice reduced hepatic LRP-1 expression which correlated with significant AD-relevant brain changes. Herein, we examined the role of hepatic LRP-1 in AD pathogenesis in APP/PS1 AD mice using two approaches to modulate hepatic LRP-1, intragastric alcohol feeding to model chronic heavy drinking shown by us to reduce hepatic LRP-1, and hepato-specific LRP-1 silencing. METHODS: Eight-month-old male APP/PS1 mice were fed ethanol or control diet intragastrically for 5 weeks (n = 7-11/group). Brain and liver Aß were assessed using immunoassays. Three important mechanisms of brain amyloidosis were investigated: hepatic LRP-1 (major peripheral Aß regulator), blood-brain barrier (BBB) function (vascular Aß regulator), and microglia (major brain Aß regulator) using immunoassays. Spatial LRP-1 gene expression in the periportal versus pericentral hepatic regions was confirmed using NanoString GeoMx Digital Spatial Profiler. Further, hepatic LRP-1 was silenced by injecting LRP-1 microRNA delivered by the adeno-associated virus 8 (AAV8) and the hepato-specific thyroxine-binding globulin (TBG) promoter to 4-month-old male APP/PS1 mice (n = 6). Control male APP/PS1 mice received control AAV8 (n = 6). Spatial memory and locomotion were assessed 12 weeks after LRP-1 silencing using Y-maze and open-field test, respectively, and brain and liver Aß were measured. RESULTS: Alcohol feeding reduced plaque-associated microglia in APP/PS1 mice brains and increased aggregated Aß (p < 0.05) by ELISA and 6E10-positive Aß load by immunostaining (p < 0.05). Increased brain Aß corresponded with a significant downregulation of hepatic LRP-1 (p < 0.01) at the protein and transcript level, primarily in pericentral hepatocytes (zone 3) where alcohol-induced injury occurs. Hepato-specific LRP-1 silencing significantly increased brain Aß and locomotion hyperactivity (p < 0.05) in APP/PS1 mice. CONCLUSION: Chronic heavy alcohol intake reduced hepatic LRP-1 expression and increased brain Aß. The hepato-specific LRP-1 silencing similarly increased brain Aß which was associated with behavioral deficits in APP/PS1 mice. Collectively, our results suggest that hepatic LRP-1 is a key regulator of brain amyloidosis in alcohol-dependent AD.

Peptide-Drug Conjugate Targeting Keratin 1 Inhibits Triple-Negative Breast Cancer in Mice.

Selective delivery of chemotherapy to the tumor site while sparing healthy cells and tissues is an attractive approach for cancer treatment. Carriers such as peptides can facilitate selective tumor targeting and payload delivery. Peptides with specific affinity for the overexpressed cell-surface receptors in cancer cells are conjugated to chemotherapy to afford peptide-drug conjugates (PDCs) that show selective uptake by cancer cells. Using a 10-mer linear peptide (WxEAAYQrFL) called 18-4 that targets and binds breast cancer cells, we designed a peptide 18-4-doxorubicin (Dox) conjugate with high specific toxicity toward triple-negative breast cancer (TNBC) MDA-MB-231 cells and 30-fold lower toxicity to normal breast MCF10A epithelial cells. Here, we elucidate the in vivo activity of this potent and tumor-selective peptide 18-4-Dox conjugate in mice bearing orthotopic MDA-MB-231 tumors. Mice treated with four weekly injections of the conjugate showed significantly lower tumor volumes compared to mice treated with free Dox at an equivalent Dox dose. Immunohistochemical (IHC) analysis of mice tissues revealed that treatment with a low dose of PDC (2.5 mg/kg of Dox equiv) reduced the expression of proliferation markers (PCNA and Ki-67) and increased apoptosis (evidenced by increased caspase-3 expression). At the same dose of free Dox (2.5 mg/kg), the expression of these markers was similar to that of saline treatment. Accordingly, significantly more Dox accumulated in tumors of conjugate-treated mice (7-fold) compared to the Dox-treated mice, while lower levels of Dox were observed in the liver, heart, and lungs of peptide-Dox conjugate-treated mice (up to 3-fold less) than Dox-treated mice. The IHC analysis of keratin 1 (K1), the receptor for peptide 18-4, revealed K1 upregulation in tumors and low levels in normal mammary fat pad and liver tissues from mice, suggesting preferential uptake of PDCs by TNBC to be K1 receptor-mediated. Taken together, our data support the use of a PDC approach to deliver chemotherapy selectively to the TNBC to inhibit tumor growth.

Valorisation of fruit peel bioactive into green synthesized silver nanoparticles to modify cellulose wrapper for shelf-life extension of packaged bread.

Fruit peels are rich source of bioactive compounds such as polyphenols, flavonoids, and antioxidants but are often discarded as waste due to limited pharmaceutical and nutraceutical applications. This study aimed to valorise pomegranate and citrus fruit peel into green synthesised silver nanoparticles (AgNPs) in order to modify cellulose-based wrapping material for prospective food packaging applications and propose an alternate and sustainable approach to replace polyethene based food packaging material. Four different concentrations of AgNO3 (0.5 mM, 1 mM, 2 mM, and 3 mM) were used for green synthesis of AgNPs from fruit peel bioactive, which were characterised followed by phytochemical analysis. Ultraviolet-Visible spectroscopy showed surface plasmon resonance at 420 nm, XRD analysis showed 2θ peak at 27.8°, 32.16°, 38.5°, 44.31°, 46.09°, 54.76°, 57.47°, 64.61° and 77.50° corresponding to (210), (122), (111), (200), (231), (142), (241), (220) and (311) plane of face centred cubic crystal structure of AgNPs. Fourier-transform infrared spectroscopy analysis of AgNPs green synthesised from pomegranate and kinnow peel extract showed a major peak at 3277, 1640 and 1250-1020 1/cm while a small peak at 2786 1/cm was observed in case of pomegranate peel extract which was negligible in AgNPs synthesized from kinnow peel extract. Particle sizes of AgNPs showed no statistically significant variance with p > 0.10 and thus, 2 mM was chosen for further experimentation and modification of cellulose based packaging material as it showed smallest average particle size. Zeta potential was observed to be nearly neutral with a partial negative strength due to presence of various phenolic compounds such as presence of gallic acid which was confirmed by ultrahigh performance liquid chromatography-photodiode array(UHPLC-PDA) detector. Thermal stability analysis of green synthesised AgNPs qualified the sterilisation conditions up to 100 °C. AgNPs green synthesized from both the peel extracts had higher polyphenolic content, antioxidant and radical scavenging activity as compared to peel extracts without treatment (p < 0.05). The cellulose based food grade packaging material was enrobed by green synthesised AgNPs. The characterisation of modified cellulose wrappers showed no significant difference in thickness of modified cellulose wrappers as compared with untreated cellulose wrapper (p > 0.42) while weight and grammage increased significantly in modified cellulose wrapper (p < 0.05). The colour values on CIE scale (L*, a* and b*) showed statistically significant increase in yellow and green colour (p < 0.05) for modified cellulose wrappers as compared to control wrapper. The oxygen permeability coefficient, water vapour permeability coefficient, water absorption capacity and water behaviour characteristics (water content, swelling degree and solubility) showed significant decrease (p < 0.05) for modified cellulose wrapper as compared to control wrapper. A uniform distribution and density of green synthesised AgNPs across cellulose wrapper matrix was observed through scanning electron microscopy (SEM) images with no significant aggregation, confirming successful enrobing and stable immobilisation of nanoparticles from cellulose matrix. A seven-day storage study of bread wrapped in modified and control cellulose wrappers showed delayed occurrence of microbial, yeast and mould count in bread packaged in modified cellulose wrappers and thus, resulting in shelf life extension of bread. The results are encouraging for the potential applications of modified cellulose wrappers to replace polyethene based food packaging.

A Small Peptide Increases Drug Delivery in Human Melanoma Cells.

Melanoma is the most fatal type of skin cancer and is notoriously resistant to chemotherapies. The response of melanoma to current treatments is difficult to predict. To combat these challenges, in this study, we utilize a small peptide to increase drug delivery to melanoma cells. A peptide library array was designed and screened using a peptide array-whole cell binding assay, which identified KK-11 as a novel human melanoma-targeting peptide. The peptide and its D-amino acid substituted analogue (VPWxEPAYQrFL or D-aa KK-11) were synthesized via a solid-phase strategy. Further studies using FITC-labeled KK-11 demonstrated dose-dependent uptake in human melanoma cells. D-aa KK-11 significantly increased the stability of the peptide, with 45.3% remaining detectable after 24 h with human serum incubation. Co-treatment of KK-11 with doxorubicin was found to significantly enhance the cytotoxicity of doxorubicin compared to doxorubicin alone, or sequential KK-11 and doxorubicin treatment. In vivo and ex vivo imaging revealed that D-aa KK-11 distributed to xenografted A375 melanoma tumors as early as 5 min and persisted up to 24 h post tail vein injection. When co-administered, D-aa KK-11 significantly enhanced the anti-tumor activity of a novel nNOS inhibitor (MAC-3-190) in an A375 human melanoma xenograft mouse model compared to MAC-3-190 treatment alone. No apparent systemic toxicities were observed. Taken together, these results suggest that KK-11 may be a promising human melanoma-targeted delivery vector for anti-melanoma cargo.

Characterization and optimization of spray dried iron and zinc nanoencapsules based on potato starch and maltodextrin.

In this study, spray drying mechanism was used to optimize the nanoencapsulation of iron and zinc using potato starch at constant proportion and variable maltodextrin and core material to keep the total solids of spray drier feed solution at 10, 20, 30 and 40% levels. Results exhibited that stable nanoencapsulates of iron and zinc were formed at 30% level with maximum in-vitro bioavailability and encapsulation efficiency of 90.68 and 89.36%, respectively. At this level the particle size and zeta potential of iron and zinc nanoencapsulates were 340.9 and 354.5 nm; 0.372 and 11.40 mV, respectively. Further, FTIR and XRD analysis of Fe 30 and Zn 30 nanoencapsulates exhibited that core material was successfully encapsulated by existence of functional groups and a semi-crystalline structure, respectively. Thus, study suggests the suitability of cheap carriers like potato starch and maltodextrin in successful encapsulation of iron and zinc indicating application potentiality in food fortification.

Keratin 1 as a cell-surface receptor in cancer.

Keratins are fibrous proteins that take part in several important cellular functions, including the formation of intermediate filaments. In addition, keratins serve as epithelial cell markers, which has made their role in cancer progression, diagnosis, and treatment an important focus of research. Keratin 1 (K1) is a type II keratin whose structure is comprised of a coiled-coil central domain flanked by flexible, glycine-rich loops in the N- and C-termini. While the structure of cytoplasmic K1 is established, the structure of cell-surface K1 is not known. Several transformed cells, such as cancerous cells and cells that have undergone oxidative stress, display increased levels of overall and/or cell-surface K1 expression. Cell-surface keratins (CSKs) may be modified or truncated, and their role is yet to be fully elucidated. Current studies suggest that CSKs are involved in receptor-mediated endocytosis and immune evasion. In this Review, we discuss findings relating to K1 structure, overexpression, and cell-surface expression in the context of utilizing CSK1 as a receptor for targeted drug delivery to cancer cells, and other strategies to develop novel treatments for cancer.

Engineering and characterization of human ß-defensin-3 and its analogues and microcin J25 peptides against Mannheimia haemolytica and bovine neutrophils.

Mannheimia haemolytica-induced bovine respiratory disease causes loss of millions of dollars to Canadian cattle industry. Current antimicrobials are proving to be ineffective and leave residues in meat. Antimicrobial peptides (AMPs) may be effective against M. haemolytica while minimizing the risk of drug residues. Cationic AMPs can kill bacteria through interactions with the anionic bacterial membrane. Human ß-Defensin 3 (HBD3) and microcin J25 (MccJ25) are AMPs with potent activity against many Gram-negative bacteria. We tested the microbicidal activity of wild-type HBD3, three HBD3 peptide analogues (28 amino acid, 20AA, and 10AA) derived from the sequence of natural HBD3, and MccJ25 in vitro against M. haemolytica. Three C-terminal analogues of HBD3 with all cysteines replaced with valines were manually synthesized using solid phase peptide synthesis. Since AMPs can act as chemoattractant we tested the chemotactic effect of HBD3, 28AA, 20AA, and 10AA peptides on bovine neutrophils in Boyden chamber. Minimum bactericidal concentration (MBC) assay showed that M. haemolytica was intermediately sensitive to HBD3, 28AA and 20AA analogues with an MBC of 50 µg/mL. The 10AA analogue had MBC 6.3 µg/mL which is likely a result of lower final inoculum size. MccJ25 didn't have significant bactericidal effect below an MBC < 100 µg/mL. Bovine neutrophils showed chemotaxis towards HBD3 and 20AA peptides (P < 0.05) but not towards 28AA analogue. Co-incubation of neutrophils with any of the peptides did not affect their chemotaxis towards N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP). The data show that these peptides are effective against M. haemolytica and are chemotactic for neutrophils in vitro.

Evaluation of a Keratin 1 Targeting Peptide-Doxorubicin Conjugate in a Mouse Model of Triple-Negative Breast Cancer.

Chemotherapy is the main treatment for triple-negative breast cancer (TNBC), a subtype of breast cancer that is aggressive with a poor prognosis. While chemotherapeutics are potent, these agents lack specificity and are equally toxic to cancer and nonmalignant cells and tissues. Targeted therapies for TNBC treatment could lead to more safe and efficacious drugs. We previously engineered a breast cancer cell targeting peptide 18-4 that specifically binds cell surface receptor keratin 1 (K1) on breast cancer cells. A conjugate of peptide 18-4 and doxorubicin (Dox) containing an acid-sensitive hydrazone linker showed specific toxicity toward TNBC cells. Here, we report the in vivo evaluation of the K1 targeting peptide-Dox conjugate (PDC) in a TNBC cell-derived xenograft mouse model. Mice treated with the conjugate show significantly improved antitumor efficacy and reduced off-target toxicity compared to mice treated with Dox or saline. After six weekly treatments, on day 35, the mice treated with PDC (2.5 mg Dox equivalent/kg) showed significant reduction (1.5 times) in tumor volume compared to mice treated with Dox (2.5 mg/kg). The mice treated with the conjugate showed significantly higher (1.4 times) levels of Dox in tumors and lower (1.3-2.2 times) levels of Dox in other organs compared to mice treated with Dox. Blood collected at 15 min showed 3.6 times higher concentration of the drug (PDC and Dox) in mice injected with PDC compared to the drug (Dox) in mice injected with Dox. The study shows that the K1 targeting PDC is a promising novel modality for treatment of TNBC, with a favorable safety profile, and warrants further investigation of K1 targeting conjugates as TNBC therapeutics.

Peptide-Drug Conjugates with Different Linkers for Cancer Therapy.

Drug conjugates are chemotherapeutic or cytotoxic agents covalently linked to targeting ligands such as an antibody or a peptide via a linker. While antibody-drug conjugates (ADCs) are now clinically established for cancer therapy, peptide-drug conjugates (PDCs) are gaining recognition as a new modality for targeted drug delivery with improved efficacy and reduced side effects for cancer treatment. The linker in a drug conjugate plays a key role in the circulation time of the conjugate and release of the drug for full activity at the target site. Herein, we highlight the main linker chemistries utilized in the design of PDCs and discuss representative examples of PDCs with different linker chemistries with the related outcome in cell and animal studies.

Effect of encapsulated vitamin E on physical, storage and retention parameters in cookies.

Microencapsulated α-tocopherol and wheat germ oil (WGO) were incorporated as WGO (5.0 ml) in liquid: WGO-L, encapsulated: WGO-E, encapsulated α-tocopherol as E1, E2 and E3 at 2.0, 3.0 and 4.0 g respectively in cookies and evaluated for physical, sensory and shelf life parameters. Spread ratio was decreased, whereas hardness was increased with encapsulated formulations and observed least in WGO-L (40.52 N) formulated cookies. During storage moisture content was observed increased (2.51-4.78%), vitamin E was retained in all formulations except WGO-L and was found maximum in E3 (4.45 mg/100 g) formulated cookies. Formulations brought the peroxide value to nil, free fatty acid development was very less, better antioxidant activity (41.1% maximum), total plate count was observed least in E3 (25 × 102 cfu/g) and good sensory acceptance of cookies up to 4 months of storage. The study concluded that encapsulated vitamin E elevated the antioxidant activity and consequently shelf life and nutritive value of cookies.

Targeting Triple Negative Breast Cancer Cells with Novel Cytotoxic Peptide-Doxorubicin Conjugates.

In this study, we have designed and synthesized two novel peptide-drug conjugates (PDCs) where the drug, doxorubicin (Dox), is linked to the peptide via a succinimidyl thioether bond or a hydrazone linker. A highly specific and proteolytically stable breast cancer cell targeting peptide (WxEAAYQrFL) is conjugated to Dox to synthesize peptide-Dox thioether (1) or hydrazone (2) conjugate. The evaluation of the stability in water, media, and human serum showed that the conjugate 1 with the succinimidyl thioether linkage is more stable compared to the acid-sensitive hydrazone containing conjugate 2. The cytotoxicity studies showed that the two PDCs were as toxic as free Dox toward the triple negative breast cancer (TNBC) cells and were 7-30 times less toxic (IC50 1.2-4.7 µM for TNBC cells versus 15-39 µM for noncancerous cells) toward the noncancerous breast cells compared to the free doxorubicin (IC50 0.35-1.5 µM for TNBC cells versus 0.24 µM for noncancerous cells). The results from the comparative study of the two PDCs suggest that both may have translational potential for TNBC treatment.

Short amylin receptor antagonist peptides improve memory deficits in Alzheimer's disease mouse model.

Recent evidence supports involvement of amylin and the amylin receptor in the pathogenesis of Alzheimer's disease (AD). We have previously shown that amylin receptor antagonist, AC253, improves spatial memory in AD mouse models. Herein, we generated and screened a peptide library and identified two short sequence amylin peptides (12-14 aa) that are proteolytically stable, brain penetrant when administered intraperitoneally, neuroprotective against Aß toxicity and restore diminished levels of hippocampal long term potentiation in AD mice. Systemic administration of the peptides for five weeks in aged 5XFAD mice improved spatial memory, reduced amyloid plaque burden, and neuroinflammation. The common residue SQELHRLQTY within the peptides is an essential sequence for preservation of the beneficial effects of the fragments that we report here and constitutes a new pharmacological target. These findings suggest that the amylin receptor antagonism may represent a novel therapy for AD.

Small Peptide Ligands for Targeting EGFR in Triple Negative Breast Cancer Cells.

The efficacy of chemotherapy for cancer treatment can be increased by targeted drug delivery to the cancer cells. This is particularly important for triple negative breast cancer (TNBC) for which chemotherapy is a major form of treatment. Here we designed and screened a library of 30 peptides starting with a previously reported epidermal growth factor receptor (EGFR) targeting peptide GE11 (YHWYGYTPQNVI). A direct peptide array-whole cell binding assay, where the peptides are conjugated to a cellulose membrane, was used to identify four peptides with enhanced binding to TNBC cells. Next, the four peptides were synthesized as FITC-labelled soluble peptides to study their direct uptake by TNBC cells using flow cytometry. The results showed that peptide analogue 22 had several fold higher uptake by the TNBC cells compared to the lead peptide GE11. The specific uptake of the peptide analogue 22 was confirmed by competition experiment using pure EGF protein. Further, peptide 22 showed dose dependent uptake by the TNBC MDA-MB-231 cells (105) with uptake saturating at around 2 µM peptide concentration. Thus, peptide 22 is a promising EGFR specific TNBC cell binding peptide that can be conjugated directly to a chemotherapeutic drug or to nanoparticles for targeted drug delivery to enhance the efficacy of chemotherapy for TNBC treatment.

Modified cantilever arrays improve sensitivity and reproducibility of nanomechanical sensing in living cells.

Mechanical signaling involved in molecular interactions lies at the heart of materials science and biological systems, but the mechanisms involved are poorly understood. Here we use nanomechanical sensors and intact human cells to provide unique insights into the signaling pathways of connectivity networks, which deliver the ability to probe cells to produce biologically relevant, quantifiable and reproducible signals. We quantify the mechanical signals from malignant cancer cells, with 10 cells per ml in 1000-fold excess of non-neoplastic human epithelial cells. Moreover, we demonstrate that a direct link between cells and molecules creates a continuous connectivity which acts like a percolating network to propagate mechanical forces over both short and long length-scales. The findings provide mechanistic insights into how cancer cells interact with one another and with their microenvironments, enabling them to invade the surrounding tissues. Further, with this system it is possible to understand how cancer clusters are able to co-ordinate their migration through narrow blood capillaries.

Modulation of Hypoxia-Induced Chemoresistance to Polymeric Micellar Cisplatin: The Effect of Ligand Modification of Micellar Carrier Versus Inhibition of the Mediators of Drug Resistance.

Hypoxia can induce chemoresistance, which is a significant clinical obstacle in cancer therapy. Here, we assessed development of hypoxia-induced chemoresistance (HICR) against free versus polymeric cisplatin micelles in a triple negative breast cancer cell line, MDA-MB-231. We then explored two strategies for the modulation of HICR against cisplatin micelles: a) the development of actively targeted micelles; and b) combination therapy with modulators of HICR in MDA-MB-231 cells. Actively targeted cisplatin micelles were prepared through surface modification of acetal-poly(ethylene oxide)-poly(α-carboxyl-ε-caprolactone) (acetal-PEO-PCCL) micelles with epidermal growth factor receptor (EGFR)-targeting peptide, GE11 (YHWYGYTPQNVI). Our results showed that hypoxia induced resistance against free and cisplatin micelles in MDA-MB-231 cells. A significant increase in micellar cisplatin uptake was observed in MDA-MB-231 cells that overexpress EGFR, following surface modification of micelles with GE11. This did not lead to increased cytotoxicity of micellar cisplatin, however. On the other hand, the addition of pharmacological inhibitors of key molecules involved in HICR in MDA-MB-231 cells, i.e., inhibitors of hypoxia inducing factor-1 (HIF-1) and signal transducer and activator of transcription 3 (STAT3), substantially enhanced the cytotoxicity of free and cisplatin micelles. The results indicated the potential benefit of combination therapy with HIF-1 and STAT3 inhibitors in overcoming HICR to free or micellar cisplatin.

Design and Evaluation of Gemini Surfactant-Based Lipoplexes Modified with Cell-Binding Peptide for Targeted Gene Therapy in Melanoma Model.

Purpose Achieving successful gene therapy requires delivery of a gene vector specifically to the targeted tissue with efficient expression and a good safety profile. The objective of this work was to develop, characterize and determine if a novel gemini surfactant-based lipoplex systems, modified with a cancer-targeting peptide p18-4, could serve this role. Methods The targeting peptide p18-4 was either chemically coupled to a gemini surfactant backbone or physically co-formulated with the lipoplexes. The influence of targeting ligand and formulation strategies on essential physicochemical properties of the lipoplexes was evaluated by dynamic light scattering and small angle X-ray scattering techniques. In vitro transfection activity and cellular toxicity of lipoplexes were assessed in a model human melanoma cell line. Results All lipoplexes zeta potential and particle size were optimal for cellular uptake and physical stability of the system. The lipoplexes adopted an inverted-hexagonal lipid arrangement. The lipoplexes modified with the peptide showed no significant changes in physicochemical properties or lipoplex assembly. The modification of the lipoplexes with the targeting peptide significantly enhanced protein expression 2-6 fold compared to non-modified lipoplexes. In addition, p18-4 modified lipoplexes significantly improved the safety of the lipoplexes. The ability of the p18-4 modified lipoplexes to selectively express the model protein was confirmed by using healthy human epidermal keratinocytes (HEKa). Conclusion The gemini surfactant-based lipoplexes modified with p18-4 peptide showed significantly higher efficiency and safety compared to the system that did not contain a cancer targeting peptide and provided evidence for their potential application to achieve targeted melanoma gene therapy.

Role of microglial amylin receptors in mediating beta amyloid (Aß)-induced inflammation.

BACKGROUND: Neuroinflammation in the brain consequent to activation of microglia is viewed as an important component of Alzheimer's disease (AD) pathology. Amyloid beta (Aß) protein is known to activate microglia and unleash an inflammatory cascade that eventually results in neuronal dysfunction and death. In this study, we sought to identify the presence of amylin receptors on human fetal and murine microglia and determine whether Aß activation of the inflammasome complex and subsequent release of cytokines is mediated through these receptors. METHODS: The presence of dimeric components of the amylin receptor (calcitonin receptor and receptor activity modifying protein 3) were first immunohistochemically identified on microglia. Purified human fetal microglial (HFM) cultures were incubated with an in vivo microglial marker, DyLight 594-conjugated tomato lectin, and loaded with the membrane-permeant green fluorescent dye, Fluo-8L-AM for measurements of intracellular calcium [Ca2+]i. HFM and BV-2 cells were primed with lipopolysaccharide and then exposed to either human amylin or soluble oligomeric Aß1-42 prior to treatment with and without the amylin receptor antagonist, AC253. Changes in the inflammasome complex, NLRP3 and caspase-1, were examined in treated cell cultures with Western blot and fluorometric assays. RT-PCR measurements were performed to assess cytokine release. Finally, in vivo studies were performed in transgenic mouse model of AD (5xFAD) to examine the effects of systemic administration of AC253 on markers of neuroinflammation in the brain. RESULTS: Acute applications of human amylin or Aß1-42 resulted in an increase in [Ca2+]i that could be blocked by the amylin receptor antagonist, AC253. Activation of the NLRP3 and caspase-1 and subsequent release of cytokines, TNFα and IL-1ß, was diminished by AC253 pretreatment of HFMs and BV2 cells. In vivo, intraperitoneal administration of AC253 resulted in a reduction in microglial markers (Iba-1 and CD68), caspase-1, TNFα, and IL-1ß. These reductions in inflammatory markers were accompanied by reduction in amyloid plaque and size in the brains of 5xFAD mice compared to controls. CONCLUSION: Microglial amylin receptors mediate Aß-evoked inflammation, and amylin receptor antagonists therefore offer an attractive therapeutic target for intervention in AD.

Traceable PEO-poly(ester) micelles for breast cancer targeting: The effect of core structure and targeting peptide on micellar tumor accumulation.

Traceable poly(ethylene oxide)-poly(ester) micelles were developed through chemical conjugation of a near-infrared (NIR) dye to the poly(ester) end by click chemistry. This strategy was tried for micelles with poly(ε-caprolactone) (PCL) or poly(α-benzyl carboxylate-ε-caprolactone) (PBCL) cores. The surface of both micelles was also modified with the breast cancer targeting peptide, P18-4. The results showed the positive contribution of PBCL over PCL core on micellar thermodynamic and kinetic stability as well as accumulation in primary orthotopic MDA-MB-231 tumors within 4-96 h following intravenous administration in mice. This was in contrast to in vitro studies where better uptake of PEO-PCL versus PEO-PBCL micelles by MDA-MB-231 cells was observed. The presence of P18-4 enhanced the in vitro cell uptake and homing of both polymeric micelles in breast tumors, but only at early time points. In conclusion, the use of developed NIR labeling technique provided means for following the fate of PEO-poly(ester) based nano-carriers in live animals. Our results showed micellar stabilization through the use of PBCL over PCL cores, to have a more significant effect in enhancing the level and duration of nano-carrier accumulation in primary breast tumors than the modification of polymeric micellar surface with breast tumor targeting peptide, P18-4.

Metabolic Study of Cancer Cells Using a pH Sensitive Hydrogel Nanofiber Light Addressable Potentiometric Sensor.

We report a simple, fast, and cost-effective approach that measures cancer cell metabolism and their response to anticancer drugs in real time. Using a Light Addressable Potentiometric Sensor integrated with pH sensitive hydrogel nanofibers (NF-LAPS), we detect localized changes in pH of the media as cancer cells consume glucose and release lactate. NF-LAPS shows a sensitivity response of 74 mV/pH for cancer cells. Cancer cells (MDA MB231) showed a response of ∼0.4 unit change in pH compared to virtually no change observed for normal cells (MCF10A). We also observed a drop in pH for the multidrug-resistant cancer cells (MDA-MB-435MDR) in the presence of doxorubicin. However, inhibition of the metabolic enzymes such as hexokinase and lactate dehydrogenase-A suggested an improvement in the efficacy of doxorubicin by decreasing the level of acidification. This approach, based on extracellular acidification, enhances our understanding of cancer cell metabolic modes and their response to chemotherapies, which will help in the development of better treatments, including choice of drugs and dosages.