Control Liposomes Research Articles

Dual Centrifugation-based Screening for pH-responsive Liposomes.

In liposomal drug delivery development, the delicate balance of membrane stability is a major challenge to prevent leakage (during shelf-life and blood circulation), and to ensure efficient payload release at the therapeutic destination. Our composite screening approach uses the processing by dual centrifugation technique to speed up the identification ofde novoformulations of intermediate membrane stability. By screening binary lipid combinations at systemically varied ratios we highlight liposomal formulations of intermediate stability, what we termed "the edge of stability", requiring moderate stimuli for destabilization. Supplementation with a pH-sensitive cholesterol derivative (to obtain acid labile liposomes) and renewed assessment with cargo load led to the discovery of three formulations with sufficient shelf-life stability, acceptable cargo retention and efficient pH-responsive cargo release in vitro. The "lead candidates" exhibited promising in cellulo uptake with increased intracellular cargo release and revealed in vivo performance advantages compared to a control liposome. Our approach filters lipid compositions on "the edge of stability" that were introduced with a pH-sensitive cholesterol derivate leading pH-responsive liposomes, out of a multidimensional parameter space. Their discovery by rational approaches would have been highly unlikely, thus highlighting the potential of our screening approach.

Abstract B051: Influence of MUC16 on the immune tumor microenvironment of PDAC

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease. In most cases, PDAC initiation results from oncogenic KRAS mutations, that act in part by directly increasing levels of a glycoprotein called Mucin-16 (MUC16). MUC16 is virtually absent in the normal pancreas, expressed on tumor cells in >65% of PDAC and is a marker of poor prognosis. The cell autonomous roles of MUC16 involve signaling through the ErbB-1/2/3 axes, and eliciting downstream AKT and GSK-3β activation to promote cell proliferation and survival. PDAC has a very interesting “immune cold” tumor microenvironment (TME) that is devoid of anti-tumor T cells and is instead flush with pro-tumor myeloid cells like macrophages. However, the role of MUC16 in influencing immune cells, particularly macrophages has not been investigated. Methods: We use immunohistochemistry to check for macrophage populations in spontaneous and orthotopic models of PDAC, KPC (LSL-Kras G12D/+ ; LSL-Trp53 R172H/+ ; Pdx-1- Cre) and a derived KPC-MUC16 knockout (KPC-M16KO). Orthotopic implantation with KPC and KPC-M16- cells was done to assess survival. In vitro cytokine screening from M16+/- cells was done to understand cytokine players in MUC16-mediated immunosuppression. Clodronate liposomes (CL) and neutral control liposomes (NL) were used to deplete macrophages in orthotopically implanted KPC and KPC-M16- tumor-bearing mice, and flow cytometry was conducted to profile macrophages and T cells in the pancreas and spleens at the experimental endpoint. Results: We show that KPC-M16KO tumors have decreased M2-like macrophages, and a modest increase in M1-like macrophages, leading to a high M1/M2 ratio suggesting reduced immune suppressed cells in the KPC-M16KO models. KPC-M16KO tumor-bearing mice showed longer survival compared to the KPC group. In addition, we detect low levels of CCL2 and Serpin-E1 in cell lines and tumor tissues from KPC-M16KO models suggesting that they could serve as potential regulators of macrophage infiltration and M2-like polarization downstream of MUC16. Siglec-E (murine homolog of human Siglec-7/9) was also downregulated in KPC-M16KO tumors, suggesting MUC16-Siglec mediated immunosuppression as another mechanism. CL administration (versus NL) resulted in significant macrophage depletion in the spleens of mice with KPC and KPC-M16KO tumors, however, such depletion was not robust in the pancreas. Immune profiling studies showed a high M1/M2 ratio in the spleen and pancreas of mice treated with CL, suggesting a tumor cell-macrophage crosstalk, that could mediate M2-like macrophage states in a macrophage-rich TME. Mice treated with CL have smaller tumors, and KPC-M16KO+CL group had the smallest tumors, indicative that dual disruption of MUC16 and macrophages lowers tumor burden. Conclusions: These results suggest that MUC16 plays a crucial role in modulating the tumor immune microenvironment by increasing the accumulation of immunosuppressive macrophages in PDAC. Citation Format: Christabelle Rajesh, Satish Sagar, Prakash Radhakrishnan. Influence of MUC16 on the immune tumor microenvironment of PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B051.

Choroid plexus immune cell response in murine hydrocephalus induced by intraventricular hemorrhage

BackgroundIntraventricular hemorrhage (IVH) and associated hydrocephalus are significant complications of intracerebral and subarachnoid hemorrhage. Despite proximity to IVH, the immune cell response at the choroid plexus (ChP) has been relatively understudied. This study employs CX3CR-1GFP mice, which marks multiple immune cell populations, and immunohistochemistry to outline that response.MethodsThis study had four parts all examining male adult CX3CR-1GFP mice. Part 1 examined naïve mice. In part 2, mice received an injection 30 µl of autologous blood into right ventricle and were euthanized at 24 h. In part 3, mice underwent intraventricular injection of saline, iron or peroxiredoxin 2 (Prx-2) and were euthanized at 24 h. In part 4, mice received intraventricular iron injection and were treated with either control or clodronate liposomes and were euthanized at 24 h. All mice underwent magnetic resonance imaging to quantify ventricular volume. The ChP immune cell response was examined by combining analysis of GFP(+) immune cells and immunofluorescence staining.ResultsIVH and intraventricular iron or Prx-2 injection in CX3CR-1GFP mice all induced ventriculomegaly and activation of ChP immune cells. There were very marked increases in the numbers of ChP epiplexus macrophages, T lymphocytes and neutrophils. Co-injection of clodronate liposomes with iron reduced the ventriculomegaly which was associated with fewer epiplexus and stromal macrophages but not reduced T lymphocytes and neutrophils.ConclusionThere is a marked immune cell response at the ChP in IVH involving epiplexus cells, T lymphocytes and neutrophils. The blood components iron and Prx-2 may play a role in eliciting that response. Reduction of ChP macrophages with clodronate liposomes reduced iron-induced ventriculomegaly suggesting that ChP macrophages may be a promising therapeutic target for managing IVH-induced hydrocephalus.

Abstract 5750: Development of prostate specific membrane antigen-targeted liposomal zinc for the treatment of prostate cancer

Abstract Introduction: Treatment choices for early-stage prostate cancer include mostly watchful waiting, radical prostatectomy, or radiation therapy, whereas for advanced cases hormone therapy followed by chemo/radiotherapy are the preferred options, despite their notable side effects. There is a need for more effective therapy with lower side effects, especially for advanced scenarios. Unlike normal epithelial prostate cells, cancerous ones show reduced zinc (Zn) levels due to ZIP1 zinc transporter downregulation. Zn deficiency allows m-aconitase to remain active, making prostate cancer cells energy efficient and grow. High levels of Zn are needed to inhibit m-aconitase, the enzyme responsible for the first reaction in the Krebs cycle. This study aims to induce energy starvation and cell death of prostate cancer cells by inhibiting m-aconitase using the prostate-specific membrane antigen (PSMA)-targeting liposomal zinc. Methods: Liposomes were prepared by standard reverse phase evaporation method with DSPC, Cholesterol and DSPE-mPEG(2000) lipids. Zinc chloride was used to load Zn+2 into liposome following passive loading method. PSMA-expressing (PC3-PIP) and PSMA-null (PC3-LUC) human prostate cancer cells were used for in vitro studies. In the cell uptake study, cells were incubated with Dil-dye labeled liposomes for 2 hours before analyzing them by microscopy and flow cytometry. For the uptake inhibition study, cells were pre-incubated with PSMA-specific peptide for 2 hours before incubation with liposomes. Cytotoxicity studies were performed with PSMA-targeting Zn-loaded liposomes in PC3-PIP cells. Results: We conjugated the PSMA-specific peptide and control peptide (ASP3) with DSPE-PEG(3400)-NHS, and used them at various amounts (1 to 4 mol% of total lipid) to prepare Zn-loaded PSMA-targeting liposomes (TL) and control liposomes (CL), respectively. The average hydrodynamic diameter of all the liposomes was 120 to 130 nm, and zeta potential ranged from -16 to -12 mV. The encapsulation efficiency and loading capacity of Zn+2 were 0.6% and 8.5%, respectively. Cellular uptake studies showed a higher accumulation of PSMA-targeting liposome (TL) compared to non-targeting liposome (CL) in PSMA-expressing PC3 cells, whereas both liposomes showed low uptake in PSMA non-expressing cell. Furthermore, pre-treatment of PSMA-expressing cells with PSMA-specific peptide reduced the uptake of TL, indicating that the uptake of TL liposome is PSMA dependent. Finally, cytotoxicity studies showed that Zn-loaded TL, induces cell death in the PSMA-expressing cells. Overall, we developed PSMA-targeting Zn-containing liposomes that selectively target and kill PSMA-expressing prostate cancer cells. Future work includes testing the effect of Zn-loaded TL on m-aconitase activity and testing of in vivo efficacy of the formulation in prostate cancer models. Citation Format: Sujan K. Mondal, Alexander L. Klibanov, Anna Moore. Development of prostate specific membrane antigen-targeted liposomal zinc for the treatment of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5750.

Anti-HER2 Immunoliposomes: Antitumor Efficacy Attributable to Targeted Delivery of Anthraquinone-Fused Enediyne.

Although natural products are essential sources of small-molecule antitumor drugs, some can exert substantial toxicities, limiting their clinical utility. Anthraquinone-fused enediyne natural products are remarkably potent antitumor drug candidates, and uncialamycin and tiancimycin (TNM) A are under development as antibody-drug conjugates. Herein, a novel drug delivery system is introduced for TNM A using anti-human epidermal growth factor receptor 2 (HER2) immunoliposomes (ILs). Trastuzumab-coated TNM A-loaded ILs (HER2-TNM A-ILs) is engineered with an average particle size of 182.8±2.1nm and a zeta potential of 1.75±0.12mV. Compared with liposomes lacking trastuzumab, HER2-TNM A-ILs exhibited selective toxicity against HER2-positive KPL-4 and SKBR3 cells. Coumarin-6, a fluorescent TNM A surrogate, is encapsulated within anti-HER2 ILs; the resultant ILs have enhanced cellular uptake in KPL-4 and SKBR3 cells when compared with control liposomes. Furthermore, ILs loaded with more Cy5.5 accumulated in KPL-4 mouse tumors. A single HER2-TNM A-IL dose (0.02mgkg-1) suppressed the growth of HER2-positive KPL-4 mouse tumors without apparent toxicity. This study not only provides a straightforward method for the effective delivery of TNM A against HER2-positive breast tumors but also underscores the potential of IL-based drug delivery systems when employing highly potent cytotoxins as payloads.

Rapid liposomal formulation for nucleolin targeting to rhabdomyosarcoma cells

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. More effective and less toxic therapies are urgently needed for high-risk patients. Peptide-guided targeted drug delivery can increase the therapeutic index of encapsulated drugs and improve patients’ well-being. To apply this strategy to RMS, we identified the peptide F3 in a screening for peptides binding to RMS cells surface. F3 binds to nucleolin, which is present on the surface of RMS cells and is abundantly expressed at the mRNA level in RMS patients’ biopsies compared to healthy tissues. We developed a rapid microfluidic formulation of F3-decorated PEGylated liposomes and remote loading of the chemotherapeutic drug vincristine. Size, surface charge, drug loading and retention of targeted and control liposomes were studied. Enhanced cellular binding and uptake were observed in three different nucleolin-positive RMS cell lines. Importantly, F3-functionalized liposomes loaded with vincristine were up to 11 times more cytotoxic than non-targeted liposomes for RMS cell lines. These results demonstrate that F3-functionalized liposomes are promising for targeted drug delivery to RMS and warrant further in vivo investigations.

Dual Affinity Nanoparticles for the Transport of Therapeutics from Carrier Cells to Target Cells under Physiological Flow Conditions.

In this study, a novel two-stage nanoparticle delivery platform was developed based on the dual functionalization of a liposome with moieties that have fundamentally different strengths of adhesion and binding kinetics. The essential concept of this system is that the nanoparticles are designed to loosely bind to the carrier cell until they come into contact with the target cell, to which they bind with greater strength. This allows the nanoparticle to be transferred from one cell to another, circulating for longer periods of time in the blood and delivering the therapeutic agent to the target circulating tumor cell. Liposomes were prepared using the lipid cake and extrusion technique, then functionalized with E-selectin (ES), anti-cell surface vimentin antibody fragments, and TRAIL via click chemistry. The binding of dual affinity (DA) liposomes was confirmed with the neutrophil-like cell line PLB985, the colorectal cancer cell line HCT116, and healthy granulocytes isolated from peripheral whole blood under physiologically relevant fluid shear stress (FSS) in a cone-and-plate viscometer. Transfer of the DA liposomes from PLB985 to HCT116 cells under FSS was greater compared to all of the control liposome formulations. Additionally, DA liposomes demonstrated enhanced apoptotic effects on HCT116 cells in whole blood under FSS, surpassing the efficacy of the ES/TRAIL liposomes previously developed by the King Lab.

Dectin-3-targeted antifungal liposomes efficiently bind and kill diverse fungal pathogens.

DectiSomes are anti-infective drug-loaded liposomes targeted to pathogenic cells by pathogen receptors including the Dectins. We have previously used C-type lectin (CTL) pathogen receptors Dectin-1, Dectin-2, and DC-SIGN to target DectiSomes to the extracellular oligoglycans surrounding diverse pathogenic fungi and kill them. Dectin-3 (also known as MCL, CLEC4D) is a CTL pathogen receptor whose known cognate ligands are partly distinct from other CTLs. We expressed and purified a truncated Dectin-3 polypeptide (DEC3) comprised of its carbohydrate recognition domain and stalk region. We prepared amphotericin B (AmB)-loaded pegylated liposomes (AmB-LLs) and coated them with this isoform of Dectin-3 (DEC3-AmB-LLs), and we prepared control liposomes coated with bovine serum albumin (BSA-AmB-LLs). DEC3-AmB-LLs bound to the exopolysaccharide matrices of Candida albicans, Rhizopus delemar (formerly known as R. oryzae), and Cryptococcus neoformans from one to several orders of magnitude more strongly than untargeted AmB-LLs or BSA-AmB-LLs. The data from our quantitative fluorescent binding assays were standardized using a CellProfiler program, AreaPipe, that was developed for this purpose. Consistent with enhanced binding, DEC3-AmB-LLs inhibited and/or killed C. albicans and R. delemar more efficiently than control liposomes and significantly reduced the effective dose of AmB. In conclusion, Dectin-3 targeting has the potential to advance our goal of building pan-antifungal DectiSomes.

Enhancing anti-tumor therapy with agmatine-cholesterol conjugate liposomes: in vitro and in vivo evidence.

In this study, we synthesized a novel compound, agmatine-cholesterol conjugate (AG-Chol), to enhance the anti-tumor activity of drug-loaded liposomes. We replaced cholesterol with AG-Chol in preparing doxorubicin hydrochloride (DOX) liposomes by using an active loading method for DOX. We assessed the physical and chemical properties of the resulting AG-Liposomes and evaluated their efficacy in vitro and in vivo. The results showed that AG-Liposomes were stable with high encapsulation efficiency. Compared with the control liposomes, AG-Liposomes exhibited a slower drug release rate in the release medium at pH 6.8. The in vitro cell experiments demonstrated that AG-Liposomes had higher tumor cell uptake rate, stronger migration inhibition rate, higher apoptosis rate, better anti-clonogenic ability, and higher lysosome escape ability than the control liposomes. In vivo distribution results demonstrate that liposomes prepared with AG-Chol instead of cholesterol can significantly enhance their tumor targeting abilities and reduce their distribution to non-targeted sites. In vivo tumor suppression experiments showed that AG-Liposomes had a higher tumor suppression rate than the control liposomes without causing apparent toxicity to normal tissues, as evidenced by histological staining. Therefore, substituting cholesterol with AG-Chol in the preparation of liposomes can result in enhanced lysosome escape, improved tumor targeting, and increased efficacy of anti-tumor drugs.

Preparation and evaluation of 111In-labeled liposomes containing phosphatidylglycerol for detection of macrophages in atherosclerotic plaques

Macrophage infiltration is a characteristic feature of atherosclerotic plaque progression. Since liposomes containing 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG) are efficiently phagocytosed by macrophages, we deduced that radiolabeled liposomes containing DSPG could potentially be used for nuclear imaging of vulnerable atherosclerotic plaques. Indium-111 (111In)-labeled liposomes containing different ratios of DSPG were developed with a high labeling efficiency. 111In-labeled liposomes with higher DSPG content showed higher uptake by macrophage-like RAW264 cells. A biodistribution study demonstrated rapid blood clearance and selective accumulation in the liver and spleen, especially in normal mice injected with 111In-labeled liposomes with higher DSPG content. Accumulation in atherosclerotic plaques was evaluated using 111In-labeled DSPG liposomes, which had the highest DSPG content among the studied liposomes. 111In-labeled DSPG liposomes accumulated in the plaques and the radioactive regions were mostly consistent with the distribution of macrophages. The target-to-non-target ratio of 111In-labeled DSPG liposomes was higher than that of 111In-labeled control liposomes without DSPG. These results suggest that 111In-labeled liposomes containing DSPG are useful for nuclear medical diagnosis of atherosclerotic plaques.

Effective mRNA Delivery by Condensation with Cationic Nanogels Incorporated into Liposomes.

The challenge in effective delivery of mRNA has been a major hurdle in their development as therapeutics. Herein, we present that the incorporation of cationic nanogels as the condensing material for mRNA into liposomes enables stable and enhanced mRNA delivery to cells in vitro. We prepared dextran-based nanogel particles, which were surface functionalized with oligoarginine peptide (DNPR9) and complexed with mRNA for incorporation into liposomes (LipoDNPR9). The use of DNPR9 with the liposomes resulted in enhanced internalization, as well as a 4-fold increase in transfection of luciferase mRNA when treated with A549 cells in vitro, compared to control liposomes. The enhancement in transfection efficiency was also observed in various cell lines while causing low cytotoxicity. The versatility of the strategy was also investigated by applying DNPR9 for mRNA condensation to ionizable lipid particles, which resulted in an ∼55% increase in transfection. The current development based on nanogel-incorporated liposomes introduces an effective platform for mRNA delivery, while the condensation strategy using DNPR9 can be widely applied for various lipid-based formulations to enhance their efficacy.

Abstract 2480: Chemotherapy increases invasive cancer stem cells in breast cancer

Abstract Background: Metastasis is the leading cause of breast cancer (BC) death. To successfully metastasize, tumor cells must have both invasive and stem-like phenotypes. BC cells that express MenaINV, an invasive isoform of the actin regulatory protein Mena, have an enhanced ability to migrate and intravasate within the primary tumor, and extravasate at secondary sites. BC cells with a stem-like phenotype can withstand anoikis during hematogenous dissemination and initiate metastatic growth. Therefore, MenaINVHigh cancer stem cells (CSCs) have high metastatic potential and can be considered the most dangerous cells in the tumor. We recently showed that MenaINV and the CSC program can be induced independently in BC cells via direct contact with tumor associated macrophages (TAMs). Though chemotherapy improves survival, recent evidence shows that for some BC patients, chemotherapy may increase metastatic dissemination, though the reasons for this are unclear. Chemotherapy causes extensive cell death and a proinflammatory cytokine surge that recruits macrophages to the primary tumor and enhances the assembly of the tumor cell dissemination portals TMEM doorways. Given that TAM-tumor cell contact induces MenaINV and the CSC program in BC cells, we hypothesized that chemotherapy promotes dissemination by increasing TAM-tumor cell contact, leading to the induction of MenaINVHigh cells, CSCs, and “double positive” MenaINVHigh CSCs. Methods: We used two models of BC (MMTV-PyMT mice bearing spontaneous mammary tumors, and HT17 patient-derived xenografts) which recapitulate metastatic triple-negative BC in mice. Mice were treated with paclitaxel (10 mg/kg, IV) every 5 days for a total of 3 treatments. Mouse subgroups received an additional treatment of liposomes with the macrophage-depleting agent clodronate (200 µL, 18.4 µM concentration, IP), or PBS control liposomes, every 2 days. Mammary tumors were evaluated for: (1) TAM density, (2) number of “single positive” cells - defined as those expressing high levels of Sox9 (CSC marker) or MenaINV (invasion marker) - and (3) the number of “double positive” MenaINVHigh CSCs (Sox9HighMenaINVHigh). Results: We found that chemotherapy increased TAM density, and this corresponded with an increase in “double positive” MenaINVHigh CSCs. Upon macrophage depletion, MenaINVHigh CSCs fell to below baseline levels regardless of chemotherapy treatment. Chemotherapy did not increase “single positive” cells. Conclusion: The induction of prometastatic MenaINVHigh CSCs during chemotherapy is macrophage dependent. The finding that chemotherapy only affected double positive cells suggests that MenaINV and the CSC program are induced together, rather than independently, by TAMs. Further work is required to determine the mechanism of this co-induction. These results lay the groundwork for novel microenvironment-based therapies to alleviate the prometastatic effects of chemotherapy in BC. Citation Format: Madeline Friedman, Camille L. Duran, Ved P. Sharma, John S. Condeelis, Maja H. Oktay, George S. Karagiannis, David Entenberg. Chemotherapy increases invasive cancer stem cells in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2480.

Regulation of protein corona on liposomes using albumin-binding peptide for targeted tumor therapy.

Nanocarriers entering the body are usually coated by plasma protein, leading to a protein "corona" easily recognized by tissues and cells. Adjusting the composition of protein coronas may be an efficient way to change the properties and behavior of nanoparticles in vivo. In this study, we modified doxorubicin-loaded liposomes (Lip/DOX) with an albumin-binding domain (ABD) to prepare nanoparticles (ABD-Lip/DOX) that can specifically bind to albumin and form albumin-based protein coronas in vivo for targeted tumor therapy. The prepared liposomes were spherical with a particle size of about 100 nm. After incubating the liposomes with rat serum, the albumin content was eight times higher on ABD-Lip than on control liposomes. ABD-Lip significantly inhibited adsorption of IgG and complement activation in rat serum in vitro, while corona-coated ABD-Lip was internalized to a significantly greater extent than corona-coated control liposomes. In addition, ABD-Lip showed longer blood circulation time, higher tumor accumulation and greater antitumor efficacy than control liposomes in mice bearing 4 T1 tumors, while both liposome formulations showed similar biocompatibility. These results confirm that adjusting the component of protein coronas around nanoparticles can improve their therapeutic efficacy.

Application of two glycosylated Lactobacillus surface layer proteins in coating cationic liposomes

The ability of isolated surface layer proteins (SLPs) to reassemble on suitable surfaces enables the application of SLPs in various fields of nanotechnology. In this work, SLPs from Lactobacillus buchneri BNCC 187,964 and L. kefir BNCC 190,565 were extracted and verified as glycosylated proteins. They were applied to coat on the surface of cationic liposomes. The absorption of the two SLPs on liposomes induced the zeta potential reduction and particle size increase. The two kinds of SLP-coated liposomes demonstrated better thermal, light and pH stability than the control liposomes. And the L. kefir SLP showed better protective effects than the L. buchneri SLP. Moreover, both of the SLPs could endow liposomes with the function of binding ferritin as observed by transmission electron microscope. Fourier transform infrared spectroscopy illustrated that the interaction between the two SLPs and liposomes was similar. The recrystallization of the two SLPs on the liposomes might drive the lipid into a higher order state and hydrogen bonds were formed between the two SLPs and the liposomes. All the findings demonstrated that L. kefir SLP and L. buchneri SLP had great potential to be explored as effective coating agents to improve the stability and function of cationic liposomes.Please check and confirm that the authors and their respective affiliations have been correctly identified and amend if necessary.Yes, all have been checked.

Macrophage cell membrane infused biomimetic liposomes for glioblastoma targeted therapy

Glioblastoma (GBM) is a highly aggressive malignant brain tumor currently without an effective treatment. Inspired by the recent advances in cell membrane biomimetic nanocarriers and by the key role of macrophages in GBM pathology, we developed macrophage membrane liposomes (MML) for GBM targeting. For the first time, it was assessed the role of macrophage polarization states in the effectiveness of these drug delivery systems. Interestingly, we observed that MML derived from M2 macrophages (M2 MML) presents higher uptake and increased delivery of the anticarcinogenic drug doxorubicin compared to M1 macrophage-derived nanocarriers (M1 MML) and control liposomes (CL). Moreover, the lowest uptake by macrophages of MML reveals promising immune escaping properties. Notably, M2 macrophages unveiled a higher expression of integrin CD49d, a crucial protein involved in the bilateral communication of macrophages with tumor cells. Therefore, our findings suggest the potential of using M2 macrophage membranes to develop novel nanocarriers targeting GBM.

Acute Toxicity Evaluation of Phosphatidylcholine Nanoliposomes Containing Nisin in Caenorhabditis elegans.

Liposomes are among the most studied nanostructures. They are effective carriers of active substances both in the clinical field, such as delivering genes and drugs, and in the food industry, such as promoting the controlled release of bioactive substances, including food preservatives. However, toxicological screenings must be performed to ensure the safety of nanoformulations. In this study, the nematode Caenorhabditis elegans was used as an alternative model to investigate the potential in vivo toxicity of nanoliposomes encapsulating the antimicrobial peptide nisin. The effects of liposomes containing nisin, control liposomes, and free nisin were evaluated through the survival rate, lethal dose (LD50), nematode development rate, and oxidative stress status by performing mutant strain, TBARS, and ROS analyses. Due to its low toxicity, it was not possible to experimentally determine the LD50 of liposomes. The survival rates of control liposomes and nisin-loaded liposomes were 94.3 and 73.6%, respectively. The LD50 of free nisin was calculated as 0.239 mg mL-1. Free nisin at a concentration of 0.2 mg mL-1 significantly affected the development of C. elegans, which was 25% smaller than the control and liposome-treated samples. A significant increase in ROS levels was observed after exposure to the highest concentrations of liposomes and free nisin, coinciding with a significant increase in catalase levels. The treatments induced lipid peroxidation as evaluated by TBARS assay. Liposome encapsulation reduces the deleterious effect on C. elegans and can be considered a nontoxic delivery system for nisin.

Modeling of the In Vitro Release Kinetics of Sonosensitive Targeted Liposomes.

Targeted liposomes triggered by ultrasound are a promising drug delivery system as they potentially improve the clinical outcomes of chemotherapy while reducing associated side effects. In this work, a comprehensive model fitting was performed for a large dataset of liposomal release profiles with seven targeting moieties (albumin, cRGD, estrone, hyaluronic acid, Herceptin, lactobionic acid, and transferrin) in addition to the control liposomes under ultrasound release protocols. Two levels of ultrasound frequencies were tested: low frequency (20 kHz) at 6.2, 9, and 10 mW/cm2 as well as high frequencies (1.07 MHz and 3 MHz) at 10.5 and 173 W/cm2. At a low frequency, Hixson-Crowell, Korsmeyer-Peppas, Gompertz, Weibull, and Lu-Hagen showed good fits to our release profiles at all three power densities. At high frequencies, the former three models reflected the best fit. These models will aid in predicting drug release profiles for future in vitro studies.

Liposomal docosahexaenoic acid halts atherosclerosis progression

Abstract Background Atherosclerosis is the main cause underlying cardiovascular disease (CVD). Docosahexaenoic acid (DHA, 22:6n-3) is a hydrophobic polyunsaturated fatty acid that exerts anti-inflammatory and antioxidant activities. However, the beneficial effects of DHA on CVD have been controversial likely due to variations in bioavailability after oral intake. Purpose In this study, we aim to investigate the potential inhibiting properties of liposomal DHA on atherosclerosis progression upon intravenous administration. Methods Four weeks old ApoE−/− and LDLr−/− mice were fed on athero-inducing high fat diet for 4 weeks and then randomly divided into two groups. The mice received either control liposomes (control group) or liposomes containing DHA (liposomal DHA treatment group) via intravenous injection, twice a week for 8 weeks while still being fed on high fat diet. At the experiment endpoint, whole aortas were collected for Oil Red O staining to quantify plaque area or for biochemical analysis. Plasma was collected for total cholesterol measurement and lipidomic analysis. Aortic roots were used for histological analysis. Results Upon intravenous injection, as shown by IVIS imaging, DHA-containing liposomes accumulated preferentially in the atherosclerotic plaques. Compared to control liposomes, liposomal DHA treatment reduced the atherosclerotic plaque area in both atherosclerosis animal models, with the total plaque area decreased by 35.8% in ApoE−/− mice, (p<0.001) and by 22.4% in LDLr−/− mice (p<0.05). Plaque composition analysis revealed that liposomal DHA treatment increased collagen content and reduced the number of macrophages and neutral lipid within the plaques, resulting in a lower plaque vulnerability index (1.095 for liposomal DHA treated group vs. 1.692 for control group, p<0.05). Among those plaque macrophages, as demonstrated by immunohistology, M2 (anti-inflammatory) macrophages accounted for 4.44% in liposomal DHA treated mice and 2.24% in control liposomes treated mice (p<0.05). In agreement with the histology results, higher mRNA expression levels of anti-inflammatory markers (IL-10, CD206 and CD163) and collagen type 1 were determined in aortic tissue after liposomal DHA treatment. Moreover, liposomal DHA did not change total cholesterol level in the blood but significantly lowered plasma levels of several species of triglycerides. In vitro experiment with bone marrow derived macrophages showed that liposomal DHA was able to suppress lipopolysaccharide-induced inflammatory response and oxidative stress. Conclusions Our findings demonstrate that incorporation of DHA in injectable liposomes is an effective way to increase the inhibitory effects of DHA on halting the progression of atherosclerosis via lowering circulating triglycerides, reducing plaque inflammation, and enhancing plaque stability. Intravenous administration of liposomal DHA may become an efficacious strategy for the treatment of atherosclerosis. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): NUSMed Seed Fund

Critical Role for Macrophages in the Developmental Programming of Pancreatic β-Cell Area in Offspring of Hypertensive Pregnancies.

Preeclampsia is a pregnancy-specific complication with long-term negative outcomes for offspring, including increased susceptibility to type 2 diabetes (T2D) in adulthood. In a rat reduced uteroplacental perfusion pressure (RUPP) model of chronic placental ischemia, maternal hypertension in conjunction with intrauterine growth restriction mimicked aspects of preeclampsia and resulted in female embryonic day 19 (e19) offspring with reduced β-cell area and increased β-cell apoptosis compared with offspring of sham pregnancies. Decreased pancreatic β-cell area persisted to postnatal day 13 (PD13) in females and could influence whether T2D developed in adulthood. Macrophage changes also occurred in islets in T2D. Therefore, we hypothesized that macrophages are crucial to reduction in pancreatic β-cell area in female offspring after chronic placental ischemia. Macrophage marker CD68 mRNA expression was significantly elevated in e19 and PD13 islets isolated from female RUPP offspring compared with sham. Postnatal injections of clodronate liposomes into female RUPP and sham offspring on PD2 and PD9 significantly depleted macrophages compared with injections of control liposomes. Depletion of macrophages rescued reduced β-cell area and increased β-cell proliferation and size in RUPP offspring. Our studies suggest that the presence of macrophages is important for reduced β-cell area in female RUPP offspring and changes in macrophages could contribute to development of T2D in adulthood.

Placental Nanoparticle Uptake-On-a-Chip: The Impact of Trophoblast Syncytialization and Shear Stress.

The placenta is a dynamic and complex organ that plays an essential role in the health and development of the fetus. Placental disorders can affect the health of both the mother and the fetus. There is currently an unmet clinical need to develop nanoparticle-based therapies to target and treat placental disorders. However, little is known about the interaction of nanoparticles (NPs) with the human placenta under biomimetic conditions. Specifically, the impact of shear stress exerted on the trophoblasts (placental epithelial cells) by the maternal blood flow, the gradual fusion of the trophoblasts along the gestation period (syncytialization), and the impact of microvilli formation on the cell uptake of NPs is not known. To this end, we designed dynamic placenta-on-a-chip models using BeWo cells to recapitulate the micro-physiological environment, and we induced different degrees of syncytialization via chemical induction with forskolin. We characterized the degree of syncytialization quantitatively by measuring beta human chorionic gonadotropin (β-hCG) secretion, as well as qualitatively by immunostaining the tight junction protein, ZO-1, and counter nuclear staining. We also characterized microvilli formation under static and dynamic conditions via F-actin staining. We used these models to measure the cell uptake of chondroitin sulfate a binding protein (CSA) conjugated and control liposomes using confocal microscopy, followed by image analysis. Interestingly, exposure of the cells to a dynamic flow of media intrinsically induced syncytialization and microvilli formation compared to static controls. Under dynamic conditions, BeWo cells produced more β-hCG in conditions that increased the cell exposure time to forskolin (p < 0.005). Our cell uptake results clearly show a combined effect of the exerted shear stress and forskolin treatment on the cell uptake of liposomes as uptake increased in forskolin exposed conditions (p < 0.05). Overall, the difference in the extent of cell uptake of liposomes among the different conditions clearly displays a need for the development of dynamic models of the placenta that consider the changes in the placental cell phenotype along the gestation period, including syncytialization, microvilli formation, and the expression of different transport and uptake receptors. Knowledge generated from this work will inform future research aiming at developing drug delivery systems targeting the placenta.