Browsing by Subject "Liposomes"

Now showing 1 - 10 of 10

Open Access
Biomedical applications of peptide nanostructures
(2016-04) Şardan Ekiz, Melis
Thesis (Ph. D.): Bilkent University, Materials Science and Nanotechnology Program, İhsan Doğramacı Bilkent University, 2016.
Open Access
Development of CpG ODN based vaccine adjuvant formulations
(Humana Press, New York, 2016) Gürsel, M.; Gürsel, İhsan; Thomas, S.
Open Access
Differential immune activation following encapsulation of immunostimulatory CpG oligodeoxynucleotide in nanoliposomes
(Elsevier, 2011) Erikçi, E.; Gursel, M.; Gürsel, T.
The immunogenicity of a vaccine formulation is closely related to the effective internalization by the innate immune cells that provide prolonged and simultaneous delivery of antigen and adjuvant to relevant antigen presenting cells. Endosome associated TLR9 recognizes microbial unmethylated CpG DNA. Clinical applications of TLR9 ligands are significantly hampered due to their pre-mature in vivo digestion and rapid clearance. Liposome encapsulation is a powerful tool to increase in vivo stability as well as enhancing internalization of its cargo to relevant immune cells. The present study established that encapsulating CpG motifs in different liposomes having different physicochemical properties altered not only encapsulation efficiency, but also the release and delivery rates that ultimately impacted in vitro and ex-vivo cytokine production rates and types. Moreover, different liposomes encapsulating CpG ODN significantly increased Th1-biased cytokines and chemokines gene transcripts Additional studies demonstrated that co-stimulatory and surface marker molecules significantly upregulated upon liposome/CpG injection. Finally, co-encapsulating model antigen ovalbumin with CpG ODN adjuvant in nanoliposomes profoundly augmented Th1 and cell mediated anti-Ova specific immune response. Collectively, this work established an unappreciated immunoregulatory property of nanoliposomes mediating immunity against protein antigen and could be harnessed to design more effective therapeutic vaccines or stand alone immunoprotective agents targeting infectious diseases, as well as cancer or allergy. © 2010 Elsevier Ltd.
Open Access
Encapsulation of two different TLR ligands into liposomes confer protective immunity and prevent tumor development
(Elsevier B.V., 2017) Bayyurt, B.; Tincer, G.; Almacioglu, K.; Alpdundar, E.; Gursel, M.; Gursel, I.
Nucleic acid-based Toll-like receptor (TLR) ligands are promising adjuvants and immunotherapeutic agents. Combination of TLR ligands potentiates immune response by providing synergistic immune activity via triggering different signaling pathways and may impact antigen dependent T-cell immune memory. However, their short circulation time due to nuclease attack hampers their clinical performance. Liposomes offer inclusion of protein and nucleic acid-based drugs with high encapsulation efficiency and drug loading. Furthermore, they protect cargo from enzymatic cleavage while providing stability, and enhancing biological activity. Herein, we aimed to develop a liposomal carrier system co-encapsulating TLR3 (polyinosinic-polycytidylic acid; poly(I:C)) and TLR9 (oligodeoxynucleotides (ODN) expressing unmethylated CpG motifs; CpG ODN) ligands as immunoadjuvants together with protein antigen. To demonstrate that this depot system not only induce synergistic innate immune activation but also boost antigen-dependent immune response, we analyzed the potency of dual ligand encapsulated liposomes in long-term cancer protection assay. Data revealed that CpG ODN and poly(I:C) co-encapsulation significantly enhanced cytokine production from spleen cells. Activation and maturation of dendritic cells as well as bactericidal potency of macrophages along with internalization capacity of ligands were elevated upon incubation with liposomes co-encapsulating CpG ODN and poly(I:C). Immunization with co-encapsulated liposomes induced OVA-specific Th1-biased immunity which persisted for eight months post-booster injection. Subsequent challenge with OVA-expressing tumor cell line, E.G7, demonstrated that mice immunized with liposomes co-encapsulating dual ligands had significantly slower tumor progression. Tumor clearance was dependent on OVA-specific cytotoxic memory T-cells. These results suggest that liposomes co-encapsulating TLR3 and TLR9 ligands and a specific cancer antigen could be developed as a preventive cancer vaccine. ï¿½ 2017 Elsevier B.V.
Open Access
In vivo applications of liposomal vaccines encapsulating single or dual pathogenassociated molecular patterns
(2017-03) Bayyurt Kocabaş, Banu
Nucleic acid-based pattern recognition receptor (PRR) agonists are promising adjuvants and immunotherapeutic agents. Combination of PRR ligands potentiates immune response by providing synergistic immune activity via triggering different signaling pathways and may impact antigen dependent T-cell immune memory. However, the duration of short circulation due to nuclease attacks is hampering their clinical performance. Liposomes enable protein and nucleic acid based compounds to have high encapsulation efficiency. Herein, we aimed to develop liposomal carrier systems that co-encapsulating single TLR9 or combinations with TLR3 or STING ligands and assess their potential as adjuvants and immunostimulatory agents in in vivo applications. Liposomal dual nucleic acid formulations induced synergistic innate immune activation, enhanced cytokine production along with internalization capacity of ligands. In anti-cancer vaccine study, CpG ODN and poly(I:C) coencapsulation significantly increased OVA-specific Th1-biased immune even after eight months post-booster injection. Challenge with OVA-expressing tumor cell line, E.G7, demonstrated that mice immunized with liposomes co-encapsulating CpG ODN and poly(I:C) had significantly slower tumor progression dependent on OVAspecific cytotoxic memory T-cells. In our second in vivo application, liposomal CDN and TLR9 therapy led to 80% remission of established melanoma tumor. Increased IgG2c/IgG1 ratio in mice treated with liposomal formulations indicating the development of antigen specific Th1-biased immunity was observed. Furthermore, along with the treatment, IFN-dual ligands into liposomes enhanced the anti-tumor activity of single ligands. In the third part, immunization with CpG ODN loaded liposomal formulations together with antigens increased antigen-specific humoral response against FMDV and Helicobacter. In addition, the liposomal CpG ODN reduced bacterial gastric colonization by antigen-dependent Th1 and Th17 immune responses after helicobacter challenging.
Open Access
Liposome encapsulation overcomes d-type and k-type CpG ODN dichotomy and induces synergistic immune activation = Lipozoma yüklenmiş D-tipi ve K-tipi ODN'lerin sinerjik immun aktivasyonu
(2014) Horuluoğlu, Begüm Han
Liposomes are one of the best candidates for the encapsulation of labile bioactive agents due to their safety and high entrapment efficiency. In human, two structurally distinct classes of CpG ODN are capable of activating different signaling pathways, leading to differential immune activation. While K-type ODN triggers plasmacytoid dendritic cells (pDCs) to mature and produce TNFα, D-type ODN leads to IRF-7 dependent IFNα secretion. Strikingly, when K-and D-type ODN are co-incubated in their free forms, K-ODN masks the D-ODN specific immune activation. Identifying proper delivery vehicles that provide both ODN types to display their superior features upon stimulation is of great clinical importance. In this study, first we investigated the synergistic effects of K- and D-ODN upon encapsulating them within five different liposome types. Then with the selected potential liposome combinations, we identified synergistic activation capacities both on human PBMCs and on mice splenocytes. In PBMC cytokine results revealed that D-ODN loaded in all five liposome types stimulated more IFNα than free D-ODN. Similarly, liposomal K-ODN triggered more TNFα than free K-ODN type. While incubation of free K and D- type ODN as expected, abrogated D-specific IFNα production from PBMC, simultaneous incubation with neutral or anionic D-ODN loaded liposomes plus cationic liposomes loaded with K-ODN significantly increased K-specific as well as D-specific effect rather than masking it (i.e. more production of TNFα and IFNα specific for K and D, respectively). This improved synergistic immune activity for both D and K ODN observed with ND+CK combination in 100% of individuals (TNFa) and 90% of individuals for IFNa. Additionally, intracellular cytokine staining findings supported improved TNFα and IFNα, from pDC population of PBMCs. Costimulatory molecule expressions and APC activation also significantly upregulated compared with free treatment. In mice contrary to ND+CK combination, sterically stabilized cationic liposome encapsulated K-ODN combined with i) neutral, ii) anionic, iii) cationic or iv) stealth encapsulated D-ODN increased IL6, IL12 and IFNγ levels, when stimulated simultaneously. Moreover, ex vivo experiments showed that cellular uptake and pro-inflammatory cytokine gene expressions significantly increased with combined liposomal formulations. This study established that by selecting proper liposome type(s) we reverse antagonistic action of K-ODN on DODN and induce a synergistic effect leading to a more robust immunostimulatory activity in both human and mice. This approach could broaden the immunotherapeutic application of these two important CpG ODN classes in clinic.
Open Access
Re-education of tumor-associated macrophages VIA TLR7/8 agonist-encapsulated liposomes
(2023-09) Tabel, Eylül
Cancer is an extremely complicated disease, and even though there have been years of effort in science to understand and find a cure, it still is one of the most terminal conditions. Lately, it is more clearly understood that the intricate wiring of the tumor microenvironment is more determinative than the intrinsic cancerous nature of tumor cells, for both disease prognosis and treatment efficiency. This environment bears many types of non-cancerous cells; such as endothelial cells, fibroblasts, and immune cells; all of which become heavily influenced by the cancer cells through their wiring of the cancer-helping niche. As proved by the success of a cancer staging approach, that utilizes a quantification of the infiltration of cytotoxic T cell populations, and which often offers a more effective staging system than traditional TNM staging for cancer, the low immunogenicity of the tumor microenvironment has been a new focus of target for kinds of therapies, including cytotoxic and immunesystem stimulation approaches One such approach is targeting tumor-associated macrophages, which are highly specialized immune cells in the tumor microenvironment responsible for many biological functions such as proliferation of cancer cells, enhancement of cancer stemness, metastasis, and a low immunogenic profile around the environment. Amongst many strategies against these cells that have extraordinary polarization and switch-of-function capabilities, polarizing them back to a tumor-fighting polarization via stimulatory molecules have yielded promising results, with the biggest obstacle of induction of a systemic immune response. Here, we have utilized liposomal encapsulation of a Toll-Like Receptor 7/8 agonist called resiquimod, in order to re-educate THP-1 macrophages that transformed into tumor-associated macrophages, back to an opposite state where they could no longer be allies of cancer cells. We have characterized the liposomes by size and morphology, and obtained a 200nm size. After we showed that this size ensured selective phagocytosis by macrophages; we loaded resiquimod molecules inside the liposomes through remote loading, with a loading efficiency of 96%. Tumorassociated macrophages that were obtained by HT-29-conditioned media incubation of THP-1 cells were characterized for their polarization state, and were re-educated with resiquimod-encapsulating liposomes. The efficiency of re-education was assessed by the reversal of tumor-associated polarization’s impact on HT-29 cells in proliferation in standard cell culture by coverage assay and in spheroid culture, cell viability by flow cytometry, metastatic capabilities by wound-healing assay, and stemness by immunocytochemistry for CD133. Overall, with a limitation of variety of tests to assess biological functions of the reeducation, we have obtained promising results for an immunotherapeutic approach of re-educating tumor-associated macrophages in colorectal cancer.
Open Access
Surface enhanced Raman spectroscopy of unilamellar liposomes loaded with silver nanoparticles
(American Scientific Publishers, 2017) Toren, P.; Tekinay, T.; Ayas S.; Dana, A.; Tunc, I.
Imaging organic molecules using surface-enhanced Raman spectroscopy (SERS) has drawn attention due to its non-invasive nature and label-free approach. The SERS approach can be used in tracking organic molecules and monitoring unique Raman spectra of the organic molecules bound to metal nanoparticles (NPs). We prepared unilamellar liposomes composed of 1,2-dipalmitoylsn-glycero-3-phosphocholine (DPPC) molecules with a diameter of around 100 nm. Electrostatic binding of silver (Ag) NPs on the surface of the unilamellar liposomes was achieved by the reduction of silver nitrate (AgNO3) which produces SERS active silver colloidal particles on the unilamellar liposome surfaces. Highly enhanced electromagnetic fields localized around neighbouring Ag NPs provide hot-spot construction around the liposomes, due to the spatial distribution of SERS enhancement in the unilamellar liposomes. It was observed that the signals fluctuate on a second time scale, presumably due to conformational change of DPPC chain and local Brownian motion of liposomal spheres.
Open Access
Therapeutic potential of an immunosuppressive oligodeoxynucleotide encapsulated within liposomes on bleomycin-induced mouse model of lung inflammation and fibrosis
(2019-05) Kılıç, Gizem
Systemic sclerosis (SSc) is an autoimmune/autoinflammatory disease with unknown etiology. It is characterized by vascular dysfunction, inflammation and disseminated fibrosis of skin or internal organs. Although its prevalence is low, development of fibrosis on internal organs and lack of a curative treatment result in high morbidity. Current therapies targeting specific symptoms such as interstitial lung disease, Raynaud’s phenomenon and pulmonary arterial hypertension are inefficient, and at best, temporarily relieves the symptoms throughout the course of the treatment. Herein, we investigated the therapeutic potential of an immunosuppressive oligodeoxynucleotide expressing TTAGGG telomeric repeats which is known as the “A151 ODN” on bleomycin-induced mouse model of systemic sclerosis. A151 ODN is the single stranded synthetic form of the telomeric repeat sequence expressed on mammalian chromosome, and it contains four repeats of “TTAGGG” motif. In order to enhance the therapeutic effectivity while protecting its digestion from nuclease activity following administration, we encapsulated A151 ODN within anionic liposomes. Since pattern recognition receptors and their signaling pathways were demonstrated to initiate inflammation in SSc, we first explored the immunosuppressive capacity of A151 ODN by analyzing in vitro cytokine productions and surface marker expression levels. Similar with the previous findings, A151 ODN was highly potent to abolish cytokine production in response to TLR9 induction. Although A151 ODN by itself was not very effective to suppress cytokine secretion following TLR1/2 and TLR4 induction, encapsulation within anionic liposomes further improved the immunosuppressive potential in response to TLR engagement. Furthermore, flow cytometry analyses revealed that A151 ODN decreased antigen presentation capacity and activation of bone-marrow derived macrophages (BMDMs) in response to TLR stimulation which was demonstrated by the reduction in levels of surface MHCII and co-stimulatory molecules as well as proteins having role on macrophage adherence and migration. A151 ODN also inhibited transcription of two major genes known to play a critical role on the development of fibrosis, TGFβ and Col1a1, from fibroblasts. Following these promising results on A151 ODN’s immunosuppressive and anti-fibrotic potential, we tested its therapeutic role on bleomycin-induced lung inflammation and fibrosis on mice which reflects different phases of systemic sclerosis. First in vivo experiment that A151 ODN was used prior to bleomycin administration revealed that A151 ODN could prevent development of systemic sclerosis by reducing immune cell recruitment into alveolar space and suppressing the secretion of inflammatory cytokines. After that, we investigated if A151 ODN could abolish established lung inflammation triggered by bleomycin instillation. For that, we treated animals with an A151 ODN either in free form or encapsulated within anionic liposomes after lung inflammation was initiated following bleomycin instillation. Data indicated that A151 ODN reduced macrophage activation marker expressions, monocyte and neutrophil infiltration into alveolar space. Moreover, suppression on immune cells activation in bronchoalveolar lavage fluid (BALF) correlated with the inhibited cytokine production. As a result of reduced inflammation, pro-fibrotic gene expressions were less in A151 ODN-treated mice. Of note, liposomal encapsulation provided reduced gene expressions while failed to further enhance the immunosuppressive potential on surface marker expression or cytokine secretion of A151 ODN. Lastly, we tested whether treatment with liposome-encapsulated A151 ODN is still effective to regress fibrosis once it has been developed; therefore, we treated mice with single injection of liposomal A151 on different time points. Unfortunately, single instillation was insufficient to decrease fibrosis and macrophage activation as well as cytokine production. Taken together, our findings indicated that liposome-encapsulated A151 ODN is very potent to attenuate the lung inflammation whereas single injection was ineffective to regress established lung fibrosis.
Open Access
TLR agonists on autoimmunity, cancer and M1/M2 macrophage polarization
(2019-07) Horuluoğlu, Begüm Han
Macrophages play an important role in the initiation of immune responses and the maintenance of immune homeostasis. Alterations in their phenotype, function and activation state have been implicated in the pathogenesis of autoimmune and inflammatory diseases. An increased M1:M2 ratio is associated with the development of several autoimmune diseases including Systematic Lupus Erythematosus (SLE), vasculitis and myositis. Previous work showed that the TLR2/1 agonist PAM3CSK4 (PAM3) could stimulate normal human monocytes to preferentially differentiate into immunosuppressive M2-like rather than inflammatory M1-like macrophages. This work seeks to investigate the ability of PAM3 to induce M2 macrophage differentiation from patient monocytes and evaluate the therapeutic efficacy of PAM3 in a murine model of lupus. Our findings revealed that patients with indicated autoimmune diseases have a significant increase in monocytes of the inflammatory subtypes coupled with a decrease in non-inflammatory classical monocytes compared to healthy controls. Additionally, in the absence of stimulant patient monocytes differentiated into more M1-like macrophages. Nevertheless, phenotypic analysis of in vitro generated macrophages revealed that, PAM3 stimulation induced M2-like macrophage differentiation without any difference from patient and healthy monocytes. Phenotypic analysis was further supported by the high endocytic abilities and secretion of anti-inflammatory cytokines instead of pro-inflammatory cytokines by PAM3 generated macrophages. Lupus-prone NZB x NZW F1 mice responded similarly to weekly PAM3 treatment. Upon PAM3 treatment the increased M1:M2 ratio, which was observed in PBS treated group, was decreased to normal levels. The increase in M2 macrophages was accompanied by decreased autoantibody and inflammatory cytokines along with an increase in anti-inflammatory cytokine production. Moreover, kidney damage was significantly suppressed and M2 macrophages were detected in the kidneys of PAM3 treated group. PAM3 treatment prolonged to survival of NZB/W significantly, at 45 weeks of age %60 of mice were still alive whereas in PBS group only %5 were. Our results indicate that, PAM3 induces immunosuppressive macrophages and thus could represent a novel approach to the therapy of autoimmune diseases. The second part of this thesis focused on enhancing the immunomodulatory effects of TLR9 ligands, CpG ODN upon encapsulation within liposomes as cancer vaccine adjuvants. Although both D and K ODN are strictly dependent on TLR9, K ODN trigger pDCs to mature and secrete TNFα while D ODN stimulate pDC to produce IFNα. When cells are incubated with a mixture of K and D ODN, K masks the activity of D. The use of both K and D ODN would be of benefit when preparing vaccine adjuvants and for immunotherapy. Our data indicate that simultaneous delivery of D ODN loaded into neutral liposomes plus K ODN loaded into cationic liposomes improved rather than masked IFNα production while continuing to support TNFα by PBMCs. Liposomal encapsulation did not alter the subcellular localization of either class of ODN, but internalization studies revealed that cationic liposome encapsulation slows and reduces the uptake of K ODN whereas neutral encapsulation of D increases their uptake by pDCs. The efficiency of K plus D liposome combinations was examined in a murine tumor vaccine model. The liposome combinations induced pronounced Th1-biased anti-OVA immunity and led to a significant reduction of B16-OVA tumors following inoculation. Our findings, demonstrate that the beneficial features of D and K ODN could be obtained simultaneously by appropriate liposomal formulation, further extending the breadth of CpG ODN-dependent immunotherapy.