HER2/neu is an immunogenic protein inducing both humoral and cell-mediated immune responses. The antigen-specific cytotoxic T lymphocytes (CTLs) are the main effector immune cells in the anti-tumor ...immunity. To induce an effective CTL specific response against P5+435 single peptide derived from rat HER2/neu oncogene, we used a liposome delivery vehicle. In vivo enhancement of liposome stability and intracytoplasmic delivery of peptides are the main strategies which elevate the liposome-mediated drug delivery. Liposomes containing high transition temperature phospholipids, such as DSPC, are stable with prolonged in vivo circulation and more accessibility to the immune system. Incorporation of DOPE phospholipid results in the effective delivery of peptide into the cytoplasm via the endocytotic pathway. To this end, the P5+435 peptide was linked to Maleimide-PEG2000-DSPE and coupled on the surface of nanoliposomes containing DSPC: DSPG: Cholesterol with/without DOPE. We observed that mice vaccinated with Lip-DOPE-P5+435 formulation had the highest number of IFN-γ- producing CTLs with the highest cytotoxic activity that consequently led to significantly smallest tumor size and prolonged survival rate in the TUBO mice model. In conclusion, our study indicated that the liposomal form of P5+435 peptide containing DOPE can be regarded as a promising prophylactic anti-cancer vaccine to generate potent antigen-specific immunity.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The present study aimed to evaluate the stability of active pharmaceutical ingredients (APIs) from different pharmacological classes in a compounded oral suspending vehicle. Oral suspensions of ...amoxicillin trihydrate (50 mg/mL), clozapine (25 mg/mL), indomethacin (5.0 mg/mL), levodopa/carbidopa (10.0/2.5 mg/mL), levothyroxine sodium (T4, 25 µg/mL), lomustine (4.0 and 10.0 mg/mL), methyldopa (25 mg/mL) and procarbazine (10.0 mg/mL) were formulated in SyrSpend® SF PH4 and the stability was monitored for up to 90 days, except for amoxicillin trihydrate, which was evaluated for 30 days only. The APIs’ stability was determined by measuring percent recovery using stability-indicating high-performance liquid chromatography (HPLC or UHPLC) or titration (amoxicillin trihydrate only). The stability of amoxicillin trihydrate, clozapine, indomethacin and levodopa/carbidopa were studied at both refrigerated (2–8 °C) and room temperature (20–25 °C). Lomustine, procarbazine, and methyldopa were studied at refrigerated temperature only. Our data demonstrated promising stability for the compounded suspensions containing various APIs, investigated in SyrSpend® SF PH4, as all APIs exhibited stability throughout the study duration and met content uniformity criteria. These findings lead to the conclusion that the tested compounded oral suspensions present a viable approach for creating personalized, age-appropriate formulations. The capacity to ensure dose consistency and stability using APIs from diverse pharmacological classes renders them suitable choices for both pediatric and geriatric patients.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The present study was aimed to develop an effective nanoliposomal vaccine delivery system with P435 HER2/neu-derived peptide conjugated to Maleimide-PEG2000-DSPE. The nanoliposome formulation ...composed of DSPC/DSPG/Chol/DOPE and monophosphoryl lipid A was used as an adjuvant. Liposomal formulations were prepared and their physical properties were characterized. Anti-tumoral efficacy of formulations was evaluated by immunization of tumor-bearing BALB/c mice and the generated immune response was studied by using ELISpot and flow cytometry analysis. The results of the study demonstrated Lip + DOPE + P535 formulation caused the lowest tumor size and the longest survival time in TUBO mice model and could make it a promising candidate in developing effective vaccines against HER2-positive breast cancers.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Modification with polyethylene glycol (PEGylation) and the use of rigid phospholipids drastically improve the pharmacokinetics of chemotherapeutics and result in more manageable or reduced ...side-effects. A major drawback is retarded cellular delivery of content, which, along with tumor heterogeneity, are the two main obstacles against tumor targeting. To enhance cellular delivery and reach a bigger area of a tumor, we designed liposomes decorated with two ligands: one for targeting tumor vasculature via a cyclic-pentapeptide containing arginine-glycine-aspartic acid (RGD), which impacts tumor independent of passive accumulation inside tumors, and one for extravascular targeting of tumor cells via a cell-penetrating peptide derived from human immunodeficiency virus type 1 transactivator of transcription (TAT). Liposomes with different ligand combinations were prepared and compared with respect to performance in targeting. Intravital imaging illustrates the heterogeneous behavior of RGD-liposomes in both intravascular and extravascular distribution, whereas TAT-liposomes exhibit a predictable extravascular localization but no intravascular targeting. Dual-ligand modification results in enhanced vascular targeting and a predictable extravascular behavior that improves the therapeutic efficacy of doxorubicin-loaded liposomes but also an augmented clearance rate of liposomes. However, the dual-modified liposome could be a great candidate for targeted delivery of non-toxic payloads or contrast agents for therapeutic or diagnostic purposes. Here we show that the combination of vascular-specific and tumor cell-specific ligands in a liposomal system is beneficial in bypassing the heterogeneous expression of tumor-specific markers.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The aim of this study is to evaluate the possible advantages of liposomes with high transition temperature (Tm) in the function of a vaccine for P5 HER2/neu-generated peptide and its adjuvant action ...to elicit CD8+ T cell response and its efficacy in TUBO in vivo tumor mice model, which over expresses the HER2/neu oncogene. P5, a hydrophobic peptide, was encapsulated in the nanoliposomes consisting of DSPC/DSPG/Chol(Tm 54°C) with a chaotropic loading system via 7M urea and described by size, zeta potential, encapsulation efficiency, and the structural stability of SDS-PAGE.
We immunized the mice for three times subcutaneously based on a two-week intervals using encapsulated peptide in the nanoliposomes, empty liposome, P5 in PBS, and PBS. Enzyme-linked immunospot assay, cytotoxicity test, and flow cytometric studies followed by the size of tumor and survival time measurements, which were done in TUBO tumor mice version.
Findings of ELISpot and flow cytometric analysis showed that immunization with Lip-p5 nanoliposomes has enhanced the antigen-specific IFN-γ response of CD8+ T cells and induced CTL response, which resulted in a smaller tumor and longer survival time. In addition to increase in amounts of IFN-γ-CD8+ T cells in a group, which was immunized with Lip-P5, our findings also revealed a Th1 shift in the group immunized with an empty liposome with reduced frequencies of IL-4-producing cells and increase of IFN-γ-producing cells.
The results indicated that simple liposomes consisting of phospholipids with high transition temperature could be an effective vaccine vehicle for tumor-associated antigens for inducing cell mediate immunity.
Tumor-associated antigen (TAA) subunit-based vaccines constitute promising tools for anticancer immunotherapy. However, a major limitation in the development of such vaccines is the poor ...immunogenicity of peptides when used alone. The aim of this study was to develop an efficient vaccine delivery system and adjuvant to enhance anti-tumor activity of a synthetic HER2/neu derived peptide (P5).
P5 peptide was encapsulated with different liposomal formulations composed of DMPC:DMPG:Chol:DOPE and loaded with monophosphoryl lipid A (MPL). All formulations were characterized for their physicochemical properties. To evaluate vaccine efficacy, BALB/c mice were first immunized with free peptide or liposomal formulations, then, inoculated with a subcutaneous injection of TUBO tumor cells. Enzyme-linked immunospot, cytotoxicity and intracellular cytokine assays, as well as tumor size and animal survival analysis, were performed to evaluate the immune responses.
The results demonstrated that P5 encapsulated into liposomal formulations was not able to induce CD8 and CD4 T cells to produce IFN-γ. That is why, a potent CTL response and antitumor immunity was not induced.
The Lip-DOPE-P5-MPL formulation in spite of using pH-sensitive lipid to direct intracellular trafficking of peptide to MHC I presentation pathway and MPL to enhance peptide adjuvanticity was interesting. The failure in inducing anti-tumor immunity may be attributed to low uptake of anionic conventional liposomes by dendritic cells (DCs) that have negative surface charge.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Highlights • Lip-DOPE-P5-MPL (DMPC/DMPG/Chol/DOPE/P5/MPL) is an effective peptide-based HER2/neu vaccine formulation. • Lip-DOPE-P5-MPL induces high IFN-γ production by CD8+ T cells intracellularly. ...• Lip-DOPE-P5-MPL induces CTL response leading to tumor growth inhibition. • Lip-DOPE-P5-MPL caused the lowest tumor size and the longest survival time in mice model of TUBO tumor.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Liposomes containing cytotoxic agents and targeted with Arg-Gly-Asp based peptides have frequently been used against αvβ3 integrin on tumor neovasculature. However, like many other ligand modified ...liposomes these preparations suffered from enhanced uptake by the reticulo endothelial system (RES) and off-targeted interaction with integrin receptors vastly expressed in normal organs causing poor biodistribution and toxic effects. Here we mainly focus on development of a RGD-modified liposomal delivery system to enhance both targeting selectivity and tumor uptake. First, sterically stabilized liposomal doxorubicin (SSLD) prepared and decorated with cRGDfK and RGDyC peptides differ in their physical properties. Stability assessments as well as in vitro and in vivo studies revealed that increasing the peptide hydrophobicity promotes the therapeutic efficacy of RGD-SSLD in a C-26 tumor model due to decreased recognition by RES and opsonization and limited off-targeted interactions. Then a novel N-methylated RGD peptide was designed and its capability in targeting integrin presenting cells was comprehensively assessed both in vitro and in vivo. RGDfN-methylC promotes the liposome internalization by HUVEC via integrin mediated endocytosis. Intravital microscopy in window chamber bearing mice illustrated the capability of RGDfN-methylC-liposomes in targeting both tumor vasculature and tumor cells in murine B16F0 and human BLM tumor models. Quantitative biodistribution in mice bearing B16F0 tumor revealed its high affinity to tumor with no considerable affinity to normal organs. Treatment by high dose of RGDfN-methylC-SSLD was found more effective than non-targeted SSLD and no toxic side effect was observed. In conclusion, the RGDfN-methylC-liposome was found promising in targeting tumor vasculature as well as other cells inside the tumor.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The aim of the present study was to improve the immunogenicity of peptide epitope vaccines using novel nanocarriers based on self-assembling materials. Several studies demonstrated that peptide ...antigens in nanoparticulate form induce stronger immune responses than their soluble forms. However, several issues such as poor loading and risk of inducing T cell anergy due to premature release of antigenic epitopes have challenged the clinical success of such systems. In the present study, we developed two vaccine delivery systems by appending a self-assembling peptide (Ac-AAVVLLLW-COOH) or a thermosensitive polymer poly(N-isopropylacrylamide (pNIPAm) to the N-terminus of different peptide antigens (OVA250–264, HPV-E743–57) to generate self-assembling peptide epitopes (SAPEs). The obtained results showed that the SAPEs were able to form nanostructures with a diameter from 20 to 200 nm. The SAPEs adjuvanted with CpG induced and expanded antigen-specific CD8+ T cells in mice. Furthermore, tumor-bearing mice vaccinated with SAPEs harboring the HPV E743–57 peptide showed a delayed tumor growth and an increased survival compared to sham-treated mice. In conclusion, self-assembling peptide based systems increase the immunogenicity of peptide epitope vaccines and therefore warrants further development toward clinical use.
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IJS, KILJ, NUK, PNG, UL, UM
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