Browsing by Subject "Liposomes."

Now showing 1 - 4 of 4

Open Access
Cell penetrating peptide amphiphile integrated liposomal systems for enhanced delivery of cargo to tumor cells
(2013) Kılınç, Murat
Liposomes have been extensively utilized as effective nanocarriers due to their enhanced solubility, higher stability and greater ability to facilitate the slow release of encapsulated drugs compared to free drug administrations. Liposomes are also preferred as drug vectors due to their non-toxic nature, biodegradability and structural resemblance to the cell membrane. However, their low internalization efficiencies pose an important challenge for their use in drug delivery applications. Internalization issues inherent in many liposomal systems can be circumvented by the use of cell penetrating peptides, which non-covalently attach on the liposome surface and greatly enhance liposomal uptake in a receptor- and charge-dependent manner. In this study, we examined the liposomal dynamics effected through the integration of an amphiphilic cell penetrating peptide into a simple liposome system. Peptide amphiphiles with a cell penetrating arginine-rich domain were incorporated into liposomal membranes formed by negatively charged dioleoylphosphoglycerol (DOPG) phospholipids in the presence of cholesterol. Throughout the present study, we sought to analyze the effect of peptide incorporation on (a) the physical characteristics, such as size, surface potential and membrane polarity, of the liposomal membrane, (b) the alterations in the encapsulation and delivery mechanisms of hydrophilic (Rhodamine B) and hydrophobic (Nile Red) drug models and (c) the enhancement of therapeutic capability in liposomes loaded with the drugs Doxorubicin-HCl and Paclitaxel. Our results revealed that the modification of liposomes by cell penetrating peptide amphiphiles results in the improvement of cargo delivery and the enhancement of the therapeutic effects of the anticancer drugs Doxurubicin and Paclitaxel.
Open Access
Enhanced immunomodulatory applications of nucleic acid encapsulating liposomes
(2009) Erikçi, Erdem
Recent studies have demonstrated that innate immune system has great ability to discriminate self from non-self through the action of innate immune receptors. The most extensively studied innate immune receptor family is the Toll-like receptors (TLRs). Endosomal/intracellular TLR3, TLR7/8 and TLR9 recognize dsRNA, ssRNA and unmethylated CpG DNA respectively. Upon activation following recognition of nucleic acids by endosomal TLRs, B cells secrete IL6, dendritic cells and macrophages secrete type I IFNs, IL12 and NK cells secrete IFNγ which yields Th1 type immune response. Modulating the immune response to mount such an immune response by TLR ligands are harnessed in medical applications such as anticancer, antiviral, antibacterial therapies, anti-allergen, as vaccine adjuvant and as immunoprotective agents. Promising clinical applications of TLR ligand nucleic acids are hampered due to their premature in vivo digestion by endonucleases and rapid clearance via serum protein absorption leading to limited stability and bioavailability. A powerful tool to overcome this problem can be achieved by encapsulating TLR ligands within liposomes, which increase in vivo stability as well as augment targeting and internalization to relevant innate immune cells. In this study we aimed to establish the most immunostimulatory liposome type encapsulating or coencapsulating CpG ODN and pIC. Five different liposomes possessing different physicochemical properties were prepared and their immunostimulatory potential when nucleic acid TLRs are loaded, were assessed. Following stimulation of splenocytes with combinations of these liposome types we have observed that neutral, anionic and stealth liposome encapsulating D-ODN, led to a dose dependent significantly higher IFNγ production over free counterpart. Stealth liposome encapsulating pIC induced both IL6 and IFNγ 10 and 250 fold respectively over free pIC. Neutral and anionic liposome coencapsulating D-ODN with pIC were very strong type 1 IFN as well as Th1 cytokine inducers both in vitro and ex vivo. Then, we immunized B6 mice with anionic liposome coencapsulating D-ODN and OVA to establish the immuno-adjuvant properties of liposome formulations in vivo. We assessed primary and secondary anti-OVA IgG subclass responses of mice. Results strongly implicated that even after primary immunization, we could obtain significantly higher anti-OVA IgG and IgG2a response over OVA mixed D-ODN group. After booster injection, 22 fold more IgG, 26 fold more IgG1 and 13 fold more IgG2a were obtained compared to free group. Our findings demonstrated that when simultaneous delivery of adjuvant (D-ODN) and antigen (OVA) within a proper depot system is given to a host, very potent antigen specific immunity is achieved. This knowledge will pave the way to design of novel effective vaccine adjuvants.
Open Access
Investigation of improved immunostimulatory activity of D and K type CpG ODNs in liposomes
(2013) Dereli, İhsan
CpG ODNs are potent immunotherapeutic agents. In human, two major classes of CpG ODNs were shown to induce differential immune activation. D ODNs are strong IFNα inducers, thus promising antiviral agents, whereas K ODNs are effective against bacterial infections. However, their effects cannot be combined. When K and D type ODNs are used simultaneously, K ODN cancels D specific effect, a phenomenon known as K and D ODN dichotomy. The prime reason for this counter acting K ODN action was subcellular compartmentalization of K type CpG ODNs upon internalization. Besides, CpG ODNs have labile nature. When investigated in clinical trials, these nucleic acid based ligands are eliminated upon administration and displayed limited bio-availability due to nuclease digestion. Hence, efforts to protect in vivo performance, and increase stability and accumulation near target cells became a crucial task. Liposome technology offers a simple and mild approach to harbor these ODNs within membrane bilayers and protect them. We also reasoned that, if we use liposomes that alter subcellular fate of K and D ODNs, we can retain both K and D effect when liposomal ODNs are co-administered and the breadth of immunotherapeutic spectrum could be improved. This thesis was designed to understand and characterize different types of CpG ODNs loaded into different liposomes and aimed to determine their activities in different in vitro and in vivo settings. Our results revealed that when two different classes of clinically important CpG ODNs were encapsulated within proper liposome types, it is possible to recapitulate both K and D type ODN effect in PBMCs. Furthermore, in a vaccine model against H. felis, although initially did not induce significantly higher anti H.felis immunity, liposomal CpG ODNs improved persisting antibody levels for extended periods compared to free counterparts. Collectively, our results demonstrate that this platform allows more effective in vivo utilization of CpG ODNs and can be formulated to develop more efficient means to combat several health problems, ranging from cancer to allergy.
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.