Calcium Enabled Remote Loading of a Weak Acid Into pH-sensitive Liposomes and Augmented Cytosolic Delivery to Cancer Cells via the Proton Sponge Effect.

While delivery of chemotherapeutics to cancer cells by nanomedicines can improve therapeutic outcomes, many fail due to the low drug loading (DL), poor cellular uptake and endosomal entrapment. This study investigated the potential to overcome these limitations using pH-sensitive liposomes (PSL) empowered by the use of calcium acetate. An acidic dinitrobenzamide mustard prodrug SN25860 was used as a model drug, with non pH-sensitive liposomes (NPSL) as a reference. Calcium acetate as a remote loading agent allowed to engineer PSL- and NPSL-SN25860 with DL of > 31.1% (w/w). The IC50 of PSL-SN25860 was 21- and 141-fold lower than NPSL and free drug, respectively. At 48 h following injection of PSL-SN25860, NPSL-SN25860 and the free drug, drug concentrations in EMT6-nfsB murine breast tumors were 56.3 µg/g, 6.76 µg/g and undetectable (< 0.015 µg/g), respectively (n = 3). Meanwhile, the ex vivo tumor clonogenic assay showed 9.1%, 19.4% and 42.7% cell survival in the respective tumors. Live-cell imaging and co-localization analysis suggested endosomal escape was accomplished by destabilization of PSL followed by release of Ca2+ in endosomes allowing induction of a proton sponge effect. Subsequent endosomal rupture was observed approximately 30 min following endocytosis of PSL containing Ca2+. Additionally, calcium in liposomes promoted internalization of both PSL and NPSL. Taken together, this study demonstrated multifaceted functions of calcium acetate in promoting drug loading into liposomes, cellular uptake, and endosomal escape of PSL for efficient cytoplasmic drug delivery. The results shed light on designing nano-platforms for cytoplasmic delivery of various therapeutics.

Mannosylation of pH-sensitive liposomes promoted cytoplasmic delivery of protein to macrophages: green fluorescent protein (GFP) performed as an endosomal escape tracer.

Conventional non-pH-sensitive liposomes for cytoplasmic delivery of protein suffer from poor efficiency. Here we investigated mannosylated pH-sensitive liposomes (MAN-PSL) for cytoplasmic delivery of protein to macrophages RAW 264.7 using PSL and non-pH-sensitive liposomes for comparison. We characterised the pH-dependent fluorescence of green fluorescent protein (GFP) and encapsulated it in liposomes as an intracellular trafficking tracer. GFP showed a reversed 'S'-shaped pH-fluorescence curve with a dramatic signal loss at acidic pH. GFP stored at 4 °C with light protection showed a half-life of 10 days (pH 5-8). The entrapment efficiency of GFP was dominated by the volume ratio of intraliposomal core to external medium for thin-film hydration. Mannosylation did not affect the pH-responsiveness of PSL. Confocal microscopy elucidated that mannosylation promoted the cellular uptake of PSL. For both these liposomes, the strongest, homogeneously distributed GFP fluorescence in the cytoplasm was found at 3 h, confirming efficient endosomal escape of GFP. Conversely, internalisation of non-pH-sensitive liposomes was slow (peaked at 12 h) and both Nile Red and GFP signals remained weak and punctuated in the cytosol. In conclusion, GFP performed as a probe for endosome escape of liposomal cargo. Mannosylation facilitated the internalisation of PSL without compromising their endosomal escape ability.

A single-shot prophylactic tumor vaccine enabled by an injectable biomembrane hydrogel.

Prophylactic tumor vaccines hold great promise against tumor occurrence. However, their clinical efficacy remains low due to inadequate activation of strong-sustainable immunity. Herein, a biomembrane hydrogel was designed as a powerful single-shot prophylactic tumor vaccine. Mannose-decorated hybrid biomembrane (MHCM) modified with oxidized sodium alginate (OSA) was designed as a gelator (O-MHCM), where the hybrid biomembrane (HCM) is a hybridization of bacterial outer membrane vesicles (OMV) and tumor cell membranes (TCM). The O-MHCM enables quick gelation subcutaneously where the cysteine protease inhibitor E64 is encapsulated in hydrogel micropores. After a single vaccination of E64@O-MHCM hydrogel, MHCM and E64 are released sustainably due to OSA moiety degradation. The MHCM enables active targeting to dendritic cells (DC) and effective DC maturation. Meanwhile, the E64 enables sufficient antigen availability for subsequent cross presentation. Ultimately, strong and sustainable T lymphocyte-mediated immunity was elicited, demonstrating a strong prophylactic effect against breast tumors. This study provides a long-lasting platform to prevent tumor occurrence, opening an innovative avenue for the design of a single-shot prophylactic tumor vaccine. STATEMENT OF SIGNIFICANCE: Developing a single-shot prophylactic tumor vaccine to elicit strong-sustainable immunity is of great interest clinically. Here, a prophylactic tumor vaccine was designed using an injectable biomembrane hydrogel for achieving strong-sustainable immunity. The mannose-tailored hybrid biomembrane was modified with oxidized sodium alginate to result in a gelator, which enabled the formation of the hydrogel after subcutaneous injection. Cysteine protease inhibitor E64 was incorporated into the micropores of the hydrogel. The hydrogel induced strong-sustainable immunity through the continuous release of active components. This was facilitated by the mannose moiety, which enabled active targeting, as well as the antigen and adjuvant function of biomembrane, and the E64-enabled suppression of antigen degradation. The biomembrane hydrogel demonstrated powerful prevention of 4T1 breast tumors. This study offers an attractive strategy for designing a single-shot prophylactic tumor vaccine.

The role of glutathione conjugation on the transcellular transport process of PEGylated liposomes across the blood brain barrier.

Notwithstanding the growing evidence of improved drug delivery efficiency to the brain by ligand modification of PEGylated liposomes, the comprehensive knowledge of their transport processes and payload across the BBB is yet to be revealed. Herein, this study sought to understand the glutathione (GSH) ligand effect on transcellular transport mechanisms of liposomes through the blood-brain barrier (BBB) by comparing PEGylated liposomes (PEG-L) and GSH PEGylated liposomes (GSH-PEG-L). Endocytosis and exocytosis of liposomes including the role of secreted extracellular vesicles (EVs) of brain endothelial cells (BECs) were assessed. Furthermore, pharmacokinetics and brain distribution analysis of gemcitabine loaded liposomes were carried out in healthy rats to ascertain the in vivo applicability. Our findings suggested that the presence of GSH increased the cellular uptake of liposomes by up to 3-fold in human brain microvascular endothelial cells depending on the dose but not in astrocytes. The cell exposure to liposomes particularly GSH-PEG-L dramatically increased the cell secretion of small and microvesicles with liposomal components, though different liposomes preferred different vesicles for exocytosis. This correlated with GSH-PEG-L transport efficiency of 4 % across the in vitro BBB model in 24 h, 1.7-fold higher than that of PEG-L (p < 0.05). In rats, while PEG-L and GSH-PEG-L showed similar pharmacokinetic profiles and prolonged circulation properties, 3.8 % of the total injected dose (ID) of gemcitabine was found in the brain of the GSH-PEG-L group at 8 h post-injection, compared with 2.8 % ID in the PEG-L group. A brain: blood concentration ratio of 1.27 ± 0.12 indicated that an active transport mechanism to cross the BBB for GSH-PEG-L. Overall, this study revealed that GSH augmented the transcellular transport efficiency of liposomes through BBB to improve targeted brain delivery by enhancing cellular uptake and vesicular exocytosis route of BECs.

A simple approach to re-engineering small extracellular vesicles to circumvent endosome entrapment.

Small extracellular vesicles (sEVs) have emerged as attractive drug delivery systems. However, the intracellular release of their cargoes is restricted. This study aimed to develop an efficient approach to re-engineer sEVs by hybridisation with pH-sensitive liposomes (PSLs) and investigate their endosome escape potential. MIA PaCa-2 cell-derived sEVs and PSLs were fused via three methods, and fusion efficiency (FE) was measured using a fluorescence resonance energy transfer assay and nanoparticle tracking analysis. Cellular uptake, intracellular trafficking, and cytotoxicity of doxorubicin-loaded vesicles (Dox@hybrids, Dox@sEVs, and Dox@PSLs) were investigated on MIA PaCa-2 cells. Among the three methods, Ca2+-mediated fusion was the simplest and led to a comparable FE with freeze-thaw method, which was significantly higher than PEG8000-mediated fusion. sEVs were more stable after hybridisation with PSLs. Confocal microscopy revealed that the hybrids internalised more efficiently than natural sEVs. While the internalised Dox@sEVs were primarily co-localised with endo/lysosomes even after 8 h, Dox from Dox@hybrids was found to escape from endosomes by 2 h and homogenously distributed in the cytosol before accumulated at nucleus, corresponding to the in vitro pH-responsive release profile. Consequently, Dox@hybrids enhanced cytotoxicity compared with Dox@sEVs, Dox@PSLs, or free drugs. Overall, the biomimetic nanosystem generated by simple Ca2+-mediated fusion was more stable and demonstrated higher efficiencies of cellular uptake and endosome escape compared to natural sEVs.

The involvement of extracellular vesicles in the transcytosis of nanoliposomes through brain endothelial cells, and the impact of liposomal pH-sensitivity.

Despite the demonstrated effectiveness of nano-materials for drug delivery to the brain, a comprehensive understanding of their transport processes across the blood brain barrier (BBB) remains undefined. This multidisciplinary study aimed to gain an insight into the transport processes across BBB, focusing on the transcytosis of liposomes and the impact of liposomal pH-sensitivity. Glutathione-PEGylated pH-sensitive (GSH-PEG-pSL) and non pH-sensitive liposomes (GSH-PEG-L) were fluorescently labelled with rhodamine-DOPE and calcein, both impermeable to biomembranes. Following exposure to brain microvascular endothelial cells (hBMECs), the key functional component of the BBB, intracellular trafficking were evaluated by confocal live-cell imaging. The exocytosed liposomes, including naturally-occurring extracellular vesicles (EVs), were collected using differential centrifugation and and characterised regarding the EV yield, morphology and EVs origin using nanoparticle tracking analysis, transmission electron microscopy and flow cytometry. The transcytosis of liposomes through a verified BBB model comprising of hBMECs monolayer was also quantified. GSH-PEG-L was initially retained in the endo-lysosomes before exocytosed while packed in EVs of different sizes (<100 â€‹nm to >1 â€‹µm) while GSH-PEG-pSL underwent endosome escape with less degree of exocytosis with more fluorescence remaining in the cytoplasm. Compared with the untreated, hBMECs treated with GSH-PEG-L increased the yield of nano-EV and medium-EV by 7.9-fold and 4.6-fold, respectively. Conversely, GSH-pSL-treated cells produced 2.9-fold more nano-EVs but 2-fold less medium-EVs than the control cells. These vesicles were CD144-positive confirming their endothelial cell-origin. GSH-PEG-L demonstrated 2-fold higher efficiencies than GSH-PEG-pSL to cross the in vitro BBB model via exocytosis. Taken together, GSH-PEG-L might utilize EV secretion pathway to achieve transcytosis across brain endothelial cells of the BBB while liposomal pH-sensitivity favors cytoplasmic delivery.

Liposomes to Augment Dialysis in Preclinical Models: A Structured Review.

In recent years, a number of groups have been investigating the use of "empty" liposomes with no drug loaded as scavengers both for exogenous intoxicants and endogenous toxic molecules. Preclinical trials have demonstrated that repurposing liposomes to sequester such compounds may prove clinically useful. The use of such "empty" liposomes in the dialysate during dialysis avoids recognition by complement surveillance, allowing high doses of liposomes to be used. The "reach" of dialysis may also be increased to molecules that are not traditionally dialysable. We aim to review the current literature in this area with the aims of increasing awareness and informing further research. A structured literature search identified thirteen papers which met the inclusion criteria. Augmenting the extraction of ammonia in hepatic failure with pH-gradient liposomes with acidic centres in peritoneal dialysis is the most studied area, with work progressing toward phase one trials. Liposomes used to augment the removal of exogenous intoxicants and protein-bound uraemic and hepatic toxins that accumulate in these organ failures and liposome-supported enzymatic dialysis have also been studied. It is conceivable that liposomes will be repurposed from the role of pharmaceutical vectors to gain further indications as clinically useful nanomedical antidotes/treatments within the next decade.

Zebularine suppressed gemcitabine-induced senescence and improved the cellular and plasma pharmacokinetics of gemcitabine, augmented by liposomal co-delivery.

Chemoresistance is a major factor driving cancer recurrence. This study investigated the potential of zebularine, a dual cytidine deaminase (CDA)/epigenetic inhibitor, to circumvent gemcitabine-resistance in pancreatic cancer using a nanomedicine co-delivery approach. The mRNA expression of key metabolic enzymes, including CDA for gemcitabine deactivation in a gemcitabine-resistant cell line Gr2000 and its parental MIA PaCa-2 was compared using quantitative reverse transcription polymerase chain reaction. A highly gemcitabine-resistant population (HRP) in Gr2000 were characterised for their growth pattern, ß-galactosidase activity (a hallmark of senescence) and chemosensitivity to zebularine after isolation. The CDA inhibition effects of zebularine on the intracellular gemcitabine accumulation and pharmacokinetics in rats when co-delivered with pH-sensitive liposomes (pSL) were investigated. Gr2000 had a 3-time upregulated mRNA expression and enzyme activity for CDA. The HRP (28% of bulk Gr2000) were predominately senescent cells which re-proliferated following a growth arrest for a week. Zebularine suppressed the regrowth of senescent cells, meanwhile enhanced cellular gemcitabine concentration by 2-fold. When co-delivered with pSL, zebularine increased cellular gemcitabine concentration by 4-fold, and extended the half-life of gemcitabine in plasma by 22-fold in rats. In conclusion, multiple mechanisms including therapy-induced senescence were identified with gemcitabine-resistance. Co-delivery of zebularine using liposomes could provide multifaceted benefits in gemcitabine therapy for pancreatic cancer treatment.

Dual or multiple drug loaded nanoparticles to target breast cancer stem cells.

Breast cancer stem(-like) cells (BCSCs) have been found to be responsible for therapeutic resistance and disease relapse. BCSCs are difficult to eradicate due to their high resistance to conventional treatments and high plasticity. Functionalised nanoparticles have been investigated as smart vehicles to transport across various barriers and increase the interaction of therapeutic agents with cancer cells, as well as BCSCs. In this review, we discuss the different characteristics of BCSCs, and challenges to tackle BCSCs at cellular and molecular levels. The mechanisms of action and physicochemical properties of the current BCSC targeting agents are also covered. We will focus on the rational design and recent advances of "Nano + Nano" or single tumour targeting nanoparticle systems loaded with dual or multiple agents to kill all cancer cells including BCSCs. These cocktail therapies include the combination of a chemotherapy agent with a BCSC-specific inhibitor, a phytochemical agent or RNA based therapy. Given the heterogeneity of breast tumour tissue, targeting both BCSCs and bulk breast cancer cells simultaneously with multiple agents holds great promise in eliminating breast cancer. The future research needs to focus on overcoming various barriers in the 'clinical translation' of BCSC-targeting nanomedicines to cure breast cancer, which requires a significant multidisciplinary effort.

Can intracellular drug delivery using hyaluronic acid functionalised pH-sensitive liposomes overcome gemcitabine resistance in pancreatic cancer?

Chemoresistance poses a major challenge in cancer treatment. This study aims to investigate whether intracellular drug delivery using hyaluronic acid (HA) functionalised pH-sensitive liposomes (HA-pSL) can circumvent gemcitabine resistance in pancreatic cancer (PC). HA-pSL were obtained by covalently conjugating HA with preformed pSL. A resistant PC cell line Gr2000 was developed by exposing MIA PaCa-2 cells to gemcitabine, and characterised for their expression of CD44, a receptor for HA, and drug transporters. Cellular uptake and intracellular trafficking of liposomes were determined by confocal microscopy and HPLC analysis of intracellular drug content. Following a pharmacokinetic study in rats, anti-tumour efficacy was compared between MIA PaCa-2 and Gr2000 xenograft mouse models. HA-pSL with an HA density of 179 µg/µmol had a larger size (152.3 vs 136.3 nm), and higher zeta potential (-46.8 vs -10.5 mV) than pSL. The sensitivity of Gr2000 to gemcitabine reduced 444 times compared to its parental cell line, despite no change to the total drug influx, as drug influx- and efflux-transporters in Gr2000 cells were simultaneously up-regulated. Both cell lines had high expression of CD44. HA facilitated cell uptake without compromising the endosome-escape ability of pSL as evidenced by confocal images and co-localization analysis of the dual-fluorescence labelled liposomes and Lysotracker. HA-pSL significantly outperformed pSL, and increased cellular drug influx by 3.6 times in MIA PaCa-2 cells, and 4.6 times in Gr2000 cells. Both liposomes improved the pharmacokinetic profile of free drug. HA-pSL treatment was superior to pSL, and resulted in 6.4 times smaller tumours (weight) in the MIA PaCa-2 xenograft models, and 3.1 smaller in the Gr2000 models compared with the free drug. Taken together, this study highlighted the use of intracellular delivery strategies (HA-CD44 interaction and endosome escape) to overcome gemcitabine resistance, however, the overall improvement was marginal and tumours still existed. Further improvement in delivery efficiency of HA-pSL to target tumours and additional manipulation of the cellular metabolism of gemcitabine are needed to tackle chemoresistance.

Pharmacokinetics and metabolism of Chf197, a ligustrazine derivative, in rats.

Ligustrazine is the most abundant and bioactive ingredient in Rhizoma Chuanxiong, a Chinese medicinal herb commonly used for the treatment of cardiovascular diseases. Chf197 is one of the structurally modified ligustrazine derivatives in a purpose of overcoming the rapid metabolism and short half-life of original. The plasma and urine pharmacokinetics of Chf197 in rats were studied after intravenous or intraperitoneal injection of Chf197 with the validated RP-HPLC method. The pharmacokinetic parameters of Chf197 injected intravenously 20 mg/kg were as follows: Cmax , 1.44 ± 0.4 mg/L; Tmax , 0.08 h; t1/2 , 3.03 ± 1.67 h; AUC, 3.85 ± 3.88 h/L; Vd , 31.66 ± 11.79L/kg; and CL, 9.29 ± 4.92 l/h/kg. Dose-dependent pharmacokinetics was observed, and a significantly higher dose-normalized AUC after intravenous administration was obtained than that after intraperitoneal administration. A possible metabolite was detected at about 3.1 min, and full-scan mass spectrum was adopted to predict its possible structure.

Angiotensin II protects cortical neurons against oxygen-glucose deprivation-induced injury in vitro.

Ischemic cerebrovascular disease is a common type of cerebrovascular disease and the leading cause of disability and mortality worldwide. Therefore, it is crucial to elucidate its pathogenesis and develop novel therapeutic strategies. This study was performed to investigate whether angiotensin (Ang) II exerts a protective effect against cerebral ischemia/reperfusion (I/R) injury in vitro. The primary cultured neurons were prepared and an I/R model was established by incubation of cortical neurons with Na2S2O4, followed by culture in fresh medium. The protective effect of Ang II and its underlying mechanisms were investigated by morphology observation, MTT assay, flow cytometry analysis and reverse transcription-polymerase chain reaction (RT-PCR). The data demonstrated that Ang II significantly ameliorated the neuronal injury caused by oxygen-glucose deprivation. Furthermore, Ang II increased cell viability through inhibiting cell apoptosis. The RT-PCR results revealed that Ang II was able to reverse the increased bax mRNA and the decreased bcl2 mRNA expression. Of note, the protective activity of Ang II may be attenuated by co-treatment with Ang II type 2 (AT2) receptor blockade (PD123319), but not Ang II type 1 (AT1) receptor blockade (valsartan). These findings suggested that Ang II exerted a protective effect against neuronal injury induced by oxygen-glucose deprivation through decreasing cell apoptosis. Therefore, Ang II may be used as a potential therapeutic target in the future.

Protective effect of ocotillol against doxorubicin­induced acute and chronic cardiac injury.

Doxorubicin (Dox) has been clinically observed to exert marked anticancer activity. However, it is severely restricted by its associated dose­dependent cardiotoxicity, which may be attenuated by decreasing the cumulative dosage via combining with a non­toxic 'sensitizer'. We previously reported that ocotillol is capable of enhancing the antitumor activity of Dox; however, the effects of ocotillol on its cardiotoxicity remain unclear. In the current study, the effects of ocotillol on the toxicity of Dox were investigated, particularly its role in cardiotoxicity. In the acute injury model, pre­administration of ocotillol prolonged the survival time. In the chronic animal model, pre­administration of ocotillol decreased the elevated levels of plasma creatine kinase (CK) and CK­MB, as well as attenuated the pathological changes that occurred. Pre­treatment with ocotillol ameliorated the decreased glutathione level and reduced the cumulated malondialdehyde in the heart tissue. In addition, pre­treatment with ocotillol restored the lowered white blood cell count. The results indicate that Dox co­treatment with ocotillol may effectively alleviate its associated toxic injury, particularly cardiotoxicity. Thus, co­administration of Dox with ocotillol may be a potential therapeutic strategy.

6r, a novel oxadiazole analogue of ethacrynic acid, exhibits antitumor activity both in vitro and in vivo by induction of cell apoptosis and S-phase arrest.

This study investigated the in vitro and in vivo antitumor effects of 5-[2,3-Dichloro-4-(2-methylene-1-oxobutyl) phenoxymethyl]-3-methyl-1,2,4- oxadiazole (6r), a novel ethacrynic acid (EA) derivative. The in vitro effect of 6r on cell proliferation of human colon, leukemia, prostate, lung, breast, ovarian and cervical tumor cell lines was assessed using MTT assay and the in vivo effect was determined with an SW620 xenografts nude mice model. The effect of 6r on expressions of GST P1-1 and apoptosis-related proteins were measured by western blotting and the effect on cell apoptosis was analysed by Hoechst 33258 nuclear staining as well as by cell surface staining of annexin V/propidium iodide. The effect on cell cycle was assessed by flow cytometry. Results showed that 6r inhibit proliferation of a range of human cancer cells in vitro and growth of SW620 tumor xenografts in vivo. The anti-proliferative effect of 6r is associated with cell apoptosis as a result of increased ratio of cellular Bax/bcl-2 expression and subsequent cytochrome-c and caspase-3 activation. Unlike EA, 6r did not show any influence on cellular GST P1-1 expression and its anti-proliferative action was associated with cell cycle arrest in G1/S-phase. In conclusion, 6r has the potential to be developed as a chemotherapeutic agent by induction of cell apoptosis but not regulating GST P1-1.

pH and temperature dual-sensitive liposome gel based on novel cleavable mPEG-Hz-CHEMS polymeric vaginal delivery system.

BACKGROUND: In this study, a pH and temperature dual-sensitive liposome gel based on a novel cleavable hydrazone-based pH-sensitive methoxy polyethylene glycol 2000-hydrazone-cholesteryl hemisuccinate (mPEG-Hz-CHEMS) polymer was used for vaginal administration. METHODS: The pH-sensitive, cleavable mPEG-Hz-CHEMS was designed as a modified pH-sensitive liposome that would selectively degrade under locally acidic vaginal conditions. The novel pH-sensitive liposome was engineered to form a thermogel at body temperature and to degrade in an acidic environment. RESULTS: A dual-sensitive liposome gel with a high encapsulation efficiency of arctigenin was formed and improved the solubility of arctigenin characterized by Fourier transform infrared spectroscopy and differential scanning calorimetry. The dual-sensitive liposome gel with a sol-gel transition at body temperature was degraded in a pH-dependent manner, and was stable for a long period of time at neutral and basic pH, but cleavable under acidic conditions (pH 5.0). Arctigenin encapsulated in a dual-sensitive liposome gel was more stable and less toxic than arctigenin loaded into pH-sensitive liposomes. In vitro drug release results indicated that dual-sensitive liposome gels showed constant release of arctigenin over 3 days, but showed sustained release of arctigenin in buffers at pH 7.4 and pH 9.0. CONCLUSION: This research has shed some light on a pH and temperature dual-sensitive liposome gel using a cleavable mPEG-Hz-CHEMS polymer for vaginal delivery.