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1.
Viruses ; 15(2)2023 02 04.
Article in English | MEDLINE | ID: mdl-36851646

ABSTRACT

Adjuvants are essential components of subunit vaccines added to enhance immune responses to antigens through immunomodulation. Very few adjuvants have been approved for human use by regulatory agencies due to safety concerns. Current subunit vaccine adjuvants approved for human use are very effective in promoting humoral immune responses but are less effective at promoting T-cell immunity. In this study, we evaluated a novel pure enantio-specific cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (R-DOTAP) as an immunomodulator for subunit vaccines capable of inducing both humoral- and cellular-mediated immunity. Using recombinant protein antigens derived from SARS-CoV2 spike or novel computationally optimized broadly reactive influenza antigen (COBRA) proteins, we demonstrated that R-DOTAP nanoparticles promoted strong cellular- and antibody-mediated immune responses in both monovalent and bivalent vaccines. R-DOTAP-based vaccines induced antigen-specific and polyfunctional CD8+ and CD4+ effector T cells and memory T cells, respectively. Antibody responses induced by R-DOTAP showed a balanced Th1/Th2 type immunity, neutralizing activity and protection of mice from challenge with live SARS-CoV2 or influenza viruses. R-DOTAP also facilitated significant dose sparing of the vaccine antigens. These studies demonstrate that R-DOTAP is an excellent immune stimulator for the production of next-generation subunit vaccines containing multiple recombinant proteins.


Subject(s)
COVID-19 , RNA, Viral , Animals , Humans , Mice , Adjuvants, Immunologic , Cations , COVID-19/prevention & control , Fatty Acids, Monounsaturated , Immunity , Lipids , SARS-CoV-2 , Vaccines, Synthetic/genetics , Antibodies, Viral/immunology
2.
Int J Pharm ; 539(1-2): 58-64, 2018 Mar 25.
Article in English | MEDLINE | ID: mdl-29366943

ABSTRACT

Synthetic membranes that are utilized in diffusion studies for topical and transdermal formulations are usually porous thin polymeric sheets for example cellulose acetate (CA) and polysulfones. In this study, the permeability of human skin was compared using two synthetic membranes: cellulose acetate and Strat-M® membrane and lipophilic and hydrophilic compounds either as saturated or formulated solutions as well as marketed dosage forms. Our data suggests that hydrophilic compounds have higher permeation in Strat-M membranes compared with lipophilic ones. High variation in permeability values, a typical property of biological membranes, was not observed with Strat-M. In addition, the permeability of Strat-M was closer to that of human skin than that of cellulose acetate (CA > Strat-M > Human skin). Our results suggest that Strat-M with little or no lot to lot variability can be applied in pilot studies of diffusion tests instead of human skin and is a better substitute than a cellulose acetate.


Subject(s)
Membranes, Artificial , Skin Absorption , Skin/metabolism , Caffeine/pharmacokinetics , Cellulose/analogs & derivatives , Diclofenac/pharmacokinetics , Humans , Hydrocortisone/pharmacokinetics , Polymers , Sulfones
3.
J Control Release ; 228: 150-158, 2016 Apr 28.
Article in English | MEDLINE | ID: mdl-26965957

ABSTRACT

Delivery of macromolecules such as siRNA into cells that reside in the basal epidermis of the skin is a major challenge due to the transport barriers that need to be overcome. siRNAs have potential therapeutic applications in various dermatological diseases such as psoriasis, atopic dermatitis, and cancer. Unfortunately, a low permeability of siRNA through the stratum corneum and epidermis has significantly limited its use for topical application. The objective of this study was to develop a topical siRNA delivery system that can permeate through the stratum corneum and viable epidermis and efficiently deposit therapeutic levels of siRNA to the basal epidermis/upper dermis where melanoma cells reside. To achieve this objective, a series of liposome compositions that contained various concentrations of edge activator in their structures were prepared and then complexed with siRNA at different ratios to generate a small library of liposome-siRNA complexes (lipoplexes) with different physicochemical properties. In this study we used melanoma as a disease model. Through use of quantitative imaging analysis, we identified the necessary design parameters for effective permeation of lipoplexes through the skin layers and deposition at the upper dermis. The ability of the formulated lipoplexes to internalize into melanoma cells, knockdown the expression of the BRAF protein and induce cell death in melanoma cells was studied by fluorescent microscopy, in-cell immunofluorescence assay and WST-1 cell proliferation assay. By providing direct quantitative and qualitative microscopy evidence, the results of this study demonstrate for the first time that the passive delivery of an edge-activated liposomal formulation can effectively carry siRNA through the stratum corneum and deposit it at the lower epidermis/upper dermis.


Subject(s)
Epidermis/metabolism , Gene Transfer Techniques , Melanoma/therapy , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/therapeutic use , RNAi Therapeutics , Cell Line, Tumor , Epidermis/pathology , Humans , Lipids/chemistry , Liposomes/chemistry , Melanoma/genetics , Melanoma/metabolism , Melanoma/pathology , Proto-Oncogene Proteins B-raf/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacokinetics , Skin Absorption
4.
Int J Pharm ; 476(1-2): 199-204, 2014 Dec 10.
Article in English | MEDLINE | ID: mdl-25263100

ABSTRACT

TXA497 is representative of a new class of guanidinomethyl biaryl compounds that exhibit potent bactericidal behavior against methicillin-resistant Staphylococcus aureus (MRSA). In this study, we compared the anti-staphylococcal, skin deposition, and skin permeation properties of TXA497 and the topical anti-MRSA antibiotic mupirocin. The results of minimum inhibitory concentration (MIC) assays revealed that TXA497 retains potent activity against MRSA that is highly resistant to mupirocin. Using Franz diffusion cells, compound deposition into human cadaver skin was evaluated, and the results showed the skin deposition of TXA497 to be significantly greater than that of mupirocin. Moreover, unlike mupirocin, TXA497 does not pass through the entire skin layer, suggesting a minimal potential for the systemic absorption of the compound upon topical administration. Additionally, antibacterial concentrations of TXA497 showed no significant toxicity to primary human keratinocytes. Given the rising levels of mupirocin resistance among MRSA populations, our results are significant in that they highlight TXA497 as a potentially useful alternative therapy for treating MRSA skin infections that are resistant to mupirocin.


Subject(s)
Anti-Bacterial Agents/pharmacology , Biphenyl Compounds/administration & dosage , Guanidines/administration & dosage , Methicillin-Resistant Staphylococcus aureus/drug effects , Mupirocin/administration & dosage , Administration, Cutaneous , Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/pharmacokinetics , Biphenyl Compounds/pharmacokinetics , Biphenyl Compounds/pharmacology , Drug Resistance, Bacterial , Guanidines/pharmacokinetics , Guanidines/pharmacology , Humans , In Vitro Techniques , Keratinocytes/metabolism , Male , Microbial Sensitivity Tests , Middle Aged , Mupirocin/pharmacokinetics , Mupirocin/pharmacology , Skin/metabolism , Skin Absorption , Staphylococcal Infections/drug therapy , Staphylococcal Infections/microbiology
5.
Biomacromolecules ; 14(6): 2033-40, 2013 Jun 10.
Article in English | MEDLINE | ID: mdl-23682625

ABSTRACT

Over the past couple of decades, the sequences of several cationic and anionic pH-responsive amphiphile fusogenic peptides (FPs) have been reported in the literature. Due to their endosome membrane disrupting activity, these peptides have been routinely used for enhancing the efficacy of drug/gene delivery systems. However, no accurate comparative study has been performed to establish the precise correlation between FP sequence and its impact on enhancing drug/gene delivery efficiency. Therefore, there has been no clear rationale for selecting one FP over another in the past, and it is still unclear which FP is the most suitable and efficient construct for use in drug/gene delivery system design. To address this shortcoming, we examined the use of a recombinant biopolymeric platform as a tool to assess the pH-dependent membrane disruption activity, cell toxicity and impact on gene transfer efficiency of the five most widely used cationic and anionic pH-responsive FPs, INF7, GALA, KALA, H5WYG, and RALA. We first developed specific expression methods for the production of five identical recombinant biopolymers that were different only in FP sequence in their structures. Through the use of physicochemical and biological assays, the biopolymers were characterized and compared in terms of DNA condensation ability, cell toxicity, pH-dependent cell membrane disruption activity, and gene transfer efficiency. Overall, our data suggests that, among the tested constructs, GALA is the most suitable pH-responsive FP for enhancing the efficiency of gene delivery systems due mostly to its efficient endosomolytic activity and negligible cell toxicity. Most importantly, this study demonstrates the application of an effective biopolymeric tool that facilitates reliable evaluation of the physicochemical and biological activities of any pH-responsive FP independent of its charge. Therefore, whether artificially designed or inspired by nature, the FPs can be screened for their efficacy with a higher degree of accuracy in the future.


Subject(s)
Biopolymers/chemistry , Hydrogen-Ion Concentration , Peptides/chemistry , Amino Acid Sequence , Molecular Sequence Data , Recombinant Proteins/chemistry
6.
Neurosci Res ; 67(1): 25-32, 2010 May.
Article in English | MEDLINE | ID: mdl-20144666

ABSTRACT

To clarify the interaction between cannabinnoid CB1 receptors and the dopaminergic system in memory processes, the effects of dopamine receptor agents on the state-dependent learning induced by the non-selective CB1/CB2 receptor agonist, WIN55,212-2 have been investigated in mice. Animals implanted with unilateral cannula at the CA1 region of the dorsal hippocampus and microinjected with WIN55,212-2 and/or dopaminergic agents, were tested using a single-trial step-down passive avoidance task. Intra-CA1 microinjections of WIN55,212-2 (0.1-1 microg/mouse) immediately after training, decreased the step-down latency, indicating an amnesic effect of the drug. The amnesia was reversed by pre-test administration of the drug, suggesting state-dependent learning by the cannabinoid. Pre-test microinjection of apomorphine, a D1/D2 dopamine receptor agonist (0.1-0.3 microg/mouse) into the CA1 region reversed the amnesia induced by post-training WIN55,212-2 (1 microg/mouse). Moreover, pre-test co-administration of apomorphine with an ineffective dose of WIN55,212-2 (0.01 microg/mouse), showed a reversion of the impairment on retention performance. Pre-test administration of the same doses of apomorphine did not show any response by itself. Pre-test intra-CA1 administration of a D1 dopamine receptor antagonist, SCH23390 (0.05-0.3 microg/mouse) or D2 dopamine receptor antagonist, sulpiride (0.125-0.5 microg/mouse) inhibited the expression of WIN55,212-2-induced state-dependent learning. Pre-test microinjection of the same doses of SCH23390 or sulpiride had no effect on WIN55,212-2-induced amnesia. Moreover, single injection of SCH23390 (0.2 and 0.3 microg/mouse) or sulpiride (0.125 microg/mouse) decreased memory retrieval. The results suggest that the dorsal hippocampal dopaminergic system participates in the modulation of WIN55,212-2-induced state-dependent learning.


Subject(s)
Avoidance Learning/drug effects , Benzoxazines/pharmacology , CA1 Region, Hippocampal/drug effects , Dopamine Antagonists/pharmacology , Morpholines/pharmacology , Naphthalenes/pharmacology , Receptor, Cannabinoid, CB1/agonists , Receptor, Cannabinoid, CB2/agonists , Animals , Apomorphine/pharmacology , Benzazepines/pharmacology , CA1 Region, Hippocampal/metabolism , Dopamine Agonists/pharmacology , Dopamine D2 Receptor Antagonists , Male , Memory/drug effects , Mice , Receptors, Dopamine D1/antagonists & inhibitors , Sulpiride/pharmacology
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