Surface-modified cationic liposomes with a matrix metalloproteinase-degradable polyethylene glycol derivative improved doxorubicin delivery in murine colon cancer.

PEGylation is a commonly used approach to prolong the blood circulation time of cationic liposomes. However, PEGylation is associated with the "PEG dilemma", which hinders binding and uptake into tumor cells. The cleavable PEG products are a possible solution to this problem. In the current research, doxorubicin-loaded cationic liposomes (Dox-CLs) surface-conjugated with a matrix metalloproteinase-2 (MMP-2)-sensitive octapeptide linker-PEG derivative were prepared and compared to non-PEGylated and PEGylated CLs in terms of size, surface charge, drug encapsulation and release, uptake, in vivo pharmacokinetics, and anticancer efficacy. It was postulated that PEG deshielding in response to the overexpressed MMP-2 in the tumor microenvironment increases the interaction of protected CLs with cellular membranes and improves their uptake by tumor cells/vasculature. MMP2-responsive Dox-CLs had particle sizes of ∼115-140 nm, surface charges of ∼+25 mV, and encapsulation efficiencies of ∼85-95%. In vitro cytotoxicity assessments showed significantly enhanced uptake and cytotoxicity of PEG-cleavable CLs compared to their non-cleavable PEG-coated counterparts or Caelyx®. Also, the chick chorioallantoic membrane assay showed great antiangiogenesis ability of Dox-CLs leading to target and prevent tumor neovascularization. Besides, in vivo studies showed an effective therapeutic efficacy of PEG-cleavable Dox-CLs in murine colorectal cancer with negligible hematological and histopathological toxicity. Altogether, our results showed that MMP2-responsive Dox-CLs could be served as a promising approach to improve tumor drug delivery and uptake.

Chemosensitization of Temozolomide-Resistant Pediatric Diffuse Midline Glioma Using Potent Nanoencapsulated Forms of a N(3)-Propargyl Analogue.

The lack of clinical response to the alkylating agent temozolomide (TMZ) in pediatric diffuse midline/intrinsic pontine glioma (DIPG) has been associated with O6-methylguanine-DNA-methyltransferase (MGMT) expression and mismatch repair deficiency. Hence, a potent N(3)-propargyl analogue (N3P) was derived, which not only evades MGMT but also remains effective in mismatch repair deficient cells. Due to the poor pharmacokinetic profile of N3P (t1/2 < 1 h) and to bypass the blood-brain barrier, we proposed convection enhanced delivery (CED) as a method of administration to decrease dose and systemic toxicity. Moreover, to enhance N3P solubility, stability, and sustained distribution in vivo, either it was incorporated into an apoferritin (AFt) nanocage or its sulfobutyl ether ß-cyclodextrin complex was loaded into nanoliposomes (Lip). The resultant AFt-N3P and Lip-N3P nanoparticles (NPs) had hydrodynamic diameters of 14 vs 93 nm, icosahedral vs spherical morphology, negative surface charge (-17 vs -34 mV), and encapsulating ∼630 vs ∼21000 N3P molecules per NP, respectively. Both NPs showed a sustained release profile and instant uptake within 1 h incubation in vitro. In comparison to the naked drug, N3P NPs demonstrated stronger anticancer efficacy against 2D TMZ-resistant DIPG cell cultures [IC50 = 14.6 (Lip-N3P) vs 32.8 µM (N3P); DIPG-IV) and (IC50 = 101.8 (AFt-N3P) vs 111.9 µM (N3P); DIPG-VI)]. Likewise, both N3P-NPs significantly (P < 0.01) inhibited 3D spheroid growth compared to the native N3P in MGMT+ DIPG-VI (100 µM) and mismatch repair deficient DIPG-XIX (50 µM) cultures. Interestingly, the potency of TMZ was remarkably enhanced when encapsulated in AFt NPs against DIPG-IV, -VI, and -XIX spheroid cultures. Dynamic PET scans of CED-administered zirconium-89 (89Zr)-labeled AFt-NPs in rats also demonstrated substantial enhancement over free 89Zr radionuclide in terms of localized distribution kinetics and retention within the brain parenchyma. Overall, both NP formulations of N3P represent promising approaches for treatment of TMZ-resistant DIPG and merit the next phase of preclinical evaluation.

Cationic liposomes formulated with a novel whole Leishmania lysate (WLL) as a vaccine for leishmaniasis in murine model.

Although there have been numerous attempts to develop a successful vaccine against leishmaniasis, based on the clinical trial in this field, no vaccine against Leishmania in routine way can be found for globally effective vaccination in human. Amongst, first generation vaccines consisting of parasite fractions or whole killed Leishmania showed more successful results in clinical trials. It seems that the main reason for the low efficacy of these vaccines is lack of a suitable adjuvant. In this study, a crude extract of detergent-solubilized L. major promastigotes as a novel developed antigen (whole Leishmania lysate (WLL)) was formulated in liposomal form. The cationic liposomes consisting of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were used to deliver WLL. Liposomes formulations containing different WLL concentrations (prepared from 103, 104, 105, 106 and 107 parasites) were prepared and characterized for particle size, surface charge, proteins, DNA and phospholipids contents. Moreover, to explore the type of immune response generated and extend of immunization, in vivo and in vitro tests including evaluation of lesion development, parasite burden in the foot and spleen, Th1 and Th2 cytokine analysis, and titration of IgG isotypes before and after the challenge were used. The maximum immunization was provided by WLL06 as depicted by the reduction of footpad swelling andparasite load, increase in anti-Leishmania IgG2a production, though no significant difference was observed between mice which received WLL05 vs WLL06. While maximum immunization was seen in WLL06 group, most of the liposomal WLL formulations induced a mixed Th1/Th2 response. Hence, a more protective immune response is expected to be induced when an immune potentiator adjuvant such as CpG ODNs would be co-deliverd in WLL liposomal formulations.

Induction of protection against leishmaniasis in susceptible BALB/c mice using simple DOTAP cationic nanoliposomes containing soluble Leishmania antigen (SLA).

A suitable adjuvant and delivery system are needed to develop an effective vaccine against leishmaniasis. To induce a Th1 type of response and protection in BALB/c mice against Leishmania major infection, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) nanoliposomes bearing an intrinsic adjuvanticity, were used as an antigen delivery system and immunoadjuvant for soluble Leishmania antigens (SLA). DOTAP liposomes containing different concentrations of SLA were prepared by using lipid film method followed by sonication. The prepared vesicles showed a diameter of about 100nm, a positive zeta potential and approximately 70% encapsulation efficiency of SLA. BALB/c mice were immunized subcutaneously (SC), three times in a 3-week interval with different concentrations of liposomal SLA (12.5, 25, and 50µg of SLA/50µl/mice), free SLA and as well as free liposome. The group of mice received 50µg of SLA in DOTAP-nanoliposomes showed a significantly (p<0.001) smaller footpad swelling and the lowest spleen and footpad parasite burden after the challenge. This group also showed the highest IFN-γ production compared to the other groups, lower IL-4 level and higher IgG2a antibody titer. Taken together, the results indicated that simple DOTAP nanoliposome containing 1µg/µl SLA are appropriate delivery systems to induce a Th1 type of immune response and protection against L. major infection in BALB/c mice.

Micro/nanoparticle adjuvants for antileishmanial vaccines: present and future trends.

Leishmania infection continues to have a major impact on public health inducing significant morbidity and mortality mostly in the poorest populations. Drug resistance, toxicity and side effects associated with expensive chemotherapeutic treatments and difficult reservoir control emphasize the need for a safe and effective vaccine which is not available yet. Although, Leishmanization (LZ) was shown to be effective against cutaneous leishmaniasis, standardization and safety are the main problems of LZ. First generation killed parasites demonstrated limited efficacy in phase 3 trials and moreover well defined molecules have not reached to phase 3 yet. Limited efficacy in vaccines against leishmaniasis is partly due to lack of an appropriate adjuvant. Hence, the use of particulate delivery systems as carriers for antigen and/or immunostimulatory adjuvants for effective delivery to the antigen-presenting cells (APCs) is a valuable strategy to enhance vaccine efficacies. Particle-based delivery systems such as emulsions, liposomes, virosomes, and polymeric microspheres have the potential for successfully delivering antigens, which can then be further improved via incorporation of additional antigenic or immustimulatory adjuvant components in or onto the particle carrier system. In this review, we have attempted to provide a list of particulate vaccine delivery systems involved in the production of candidate leishmaniasis vaccines and introduced some potentially useful vaccine delivery systems for leishmaniasis in future experiments. In conclusion, combination vaccines (adjuvant systems) composed of candidate antigens and more importantly well-developed particulate delivery systems, such as lipid-based particles containing immunostimulatory adjuvants, have a chance to succeed as antileishmanial vaccines.

Cationic liposomes containing soluble Leishmania antigens (SLA) plus CpG ODNs induce protection against murine model of leishmaniasis.

Development of an effective vaccine against leishmaniasis is possible due to the fact that individuals cured from cutaneous leishmaniasis (CL) are protected from further infection. First generation Leishmania vaccines consisting of whole killed parasites reached to phase 3 clinical trials but failed to show enough efficacies mainly due to the lack of an appropriate adjuvant. In this study, an efficient liposomal protein-based vaccine against Leishmania major infection was developed using soluble Leishmania antigens (SLA) as a first generation vaccine and cytidine phosphate guanosine oligodeoxynucleotides (CpG ODNs) as an immunostimulatory adjuvant. 1, 2-Dioleoyl-3-trimethylammonium-propane was used as a cationic lipid to prepare the liposomes due to its intrinsic adjuvanticity. BALB/c mice were immunized subcutaneously (SC), three times in 2-week intervals, with Lip-SLA-CpG, Lip-SLA, SLA + CpG, SLA, or HEPES buffer. As criteria for protection, footpad swelling at the site of challenge and spleen parasite loads were assessed, and the immune responses were evaluated by determination of IFN-γ and IL-4 levels of cultured splenocytes, and IgG subtypes. The group of mice that received Lip-SLA-CpG showed a significantly smaller footpad swelling, lower spleen parasite burden, higher IgG2a antibody, and lower IL-4 level compared to the control groups. It is concluded that cationic liposomes containing SLA and CpG ODNs are appropriate to induce Th1 type of immune response and protection against leishmaniasis.