18 research outputs found
Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo
While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential
Koletsüstokiniini seotus kroonilise valu mehhanismidega ja endogeense valuvastase süsteemiga
Current thesis explored the chronic pain phenotype of the CCK2 receptor deficient mice and tried to match some neurochemical correlates to the phenotype. First, we established that the CCK2-/- mice displayed hyposensitivity in the von Frey mechanical sensitivity testing model. The involvement of CCK-opioid reciprocal regulation was confirmed in this effect, because the hyposensitivity phenotype was reversed by administrating naloxone (0.1…10 mg/kg), antagonist of opioid receptors and, on the other hand, hyposensitivity was induced in the wild-type mice by administering L-365260 (0.01…1 mg/kg), a CCK2 receptor antagonist. Second, we established insensitivity to neuropathic pain in the CCK2-/- mice – only minor hypersensitivity was seen in these mice after conducting chronic constriction injury, as opposed to development of extensive hypersensitivity in wild-type animals. Gene expression analysis demonstrated disturbances in opioid peptide or opioid receptor gene expression in the mutant mice. We conclude that disruption of the CCK signal through the CCK2 receptor upregulates endogenous opioids to a degree that no apparent neuropathic pain symptoms develop.
In the second part of the thesis we explored gene expression changes in the CCK2-/- mice in brainstem and midbrain, following induction of neuropathic pain, using Affymetrix mouse gene expression chips. We established strong activation of MAPK pathway as a response to peripheral nerve injury, regardless of the genotype in both CNS areas. We did not establish strong genotype x peripheral nerve injury interactions, the most interesting genotype effect (ANOVA genotype main effect) being Tlr4, a lipopolysaccharide recognizing gene. This again supports immune activation in the brainstem and midbrain following peripheral nerve injury and suggests that CCK2 receptor is implicated in interactions between immune system activation and CNS.
Third, we studied stress-induced analgesia and the role of CCK and endocannabinoids. Stress-analgesia is an important manifestation of adaptational mechanisms. In addition, studying stress-analgesia provides us with information about endogenous analgesic mechanisms that could be used in clinical practice. We used electric foot-shock (0.6 mA, 3 min) as a stress model and measured pre-stress and post-stress analgesia levels with tail flick. We established that the CCK2-/- mice developed smaller analgesia to the same amount of stress. This implies that eliminating CCK2 signal disrupts some endogenous analgesic mechanisms. We administered opioid antagonist naloxone (0.01…10 mg/kg) and cannabinoid CB1 receptor antagonist rimonabant (0.1…3 mg/kg) and tested the effect on stress-induced analgesia. Naloxone blocked the analgesia in both CCK2-/- and wild-type mice. In contrast, rimonabant blocked the stress-analgesia only in wild-type animals and did not modify the knockout response. This result is in good agreement with subsequent gene expression profiling that demonstrated extensive activation of cannabinoid system in the wild-type mice in response to stress. This stress-related cannabinoid activation was largely absent in the mutant mice. We conclude that the signalling of CCK through CCK2 receptor is necessary for the stress-related endocannabinoid activation, as the CCK2-/- mice lack the endocannabinoid component of the stress-analgesia.
Finally, we analysed the previously described Affymetrix gene chip data from the neuropathic animals from a different angle. Studying only the genotype effect (i.e. omitting the nerve injury factor) we observed that majority of the genotype differences (i.e. genes differentially expressed in CCK2-/- animals) mapped in close proximity (40 Mbp area) to the CCK2 gene in the mouse genome. Almost all genes with changed expression between the genotypes were in knockout-low direction. The genes clustered together by genomic location, but not by gene function. We confirmed that these genes were of 129sv parent line origin that were not lost even through extensive backcrossing to the C57BL/6 background. We conclude that mouse gene knockouts generated to the mixed background is not as clean model as historically thought and the observed phenotype of these mice may not be the result of only disruption of the single target gene.
Käesoleva dissertatsiooni käigus uuriti CCK2 retseptori puudulike (CCK2–/–) hiirte kroonilise valu fenotüüpi ja üritati sellele fenotüübile leida ka neurokeemilisi korrelaate. Esiteks leidsime, et CCK2–/– hiirtel esines von Frey mehaanilise tundlikkuse testimise mudelis hüposensitiivsus. Selle leiu põhjuseks leiti olevat CCK ja opioidide vastastikune regulatsioon, sest hüposensitiivsuse fenotüübi sai blokeerida opioidi retseptorite antagonisti naloksooni (0.1…10 mg/kg) manustamisega ja teisalt sai metsiktüüpi loomadel esile kutsuda hüposensitiivsuse CCK2 antagonisti L-365260 (0.01…1 mg/kg) manustamisega. Teiseks kirjeldasime CCK2–/– hiirtel tundetust neuropaatilise valu suhtes, sest pärast kroonilise kompressiooni vigastust (chronic constriction injury) tekkis neil loomadel ainult minimaalne ülitundlikkus, vastandina metsiktüüpi loomadele, kellel tekkis väga tugev ülitundlikkus. Geeniekspressioon-analüüs näitas mutantsetel hiirtel häireid opioidsete peptiidide ja opioidiretseptorite geeniekspressioonis. Sellest järeldame, et CCK signaali häirimine läbi CCK2 retseptori võimendab opioidisüsteemi sellisel määral, et nähtavaid neuropaatilise valu sümptomeid ei teki.
Käesoleva töö teises osas uurisime kroonilise kompressiooni vigastuse tekitamise järgselt CCK2–/– hiirte geeniekspressiooni muutusi ajutüves ja keskajus, kasutades Affymetrix'i hiire geeniekspressiooni kiipe. Leidsime, et vastuseks närvivigastusele, sõltumata genotüübist tekib tugev MAPK raja aktivatsioon mõlemas KNS piirkonnas. Me ei leidnud tugevaid genotüübi x perifeerse närvi vigastuse interaktsioone. Kõige huvitavam genotüübist sõltuv (genotüübi ANOVA peaefekt) kandidaatgeen oli Tlr4, lipopolüsahhariidile reageeriv geen. Ka see tulemus näitab perifeerse närvi vigastuse järgset immuunaktivatsiooni ajutüves ja keskajus ja lubab oletada, et CCK2 retseptor on seotud immuunsüsteemi aktivatsiooni ja KNS vaheliste interaktsioonidega.
Kolmandaks uurisime stress-analgeesiat ja CCK ja endokannabinoidide rolli selles. Stress-analgeesia on oluline adaptatsioonimehhanismide näitaja. Lisaks sellele võimaldab stress-analgeesia uurimine endogeensetest analgeetilistest mehhanismidest rohkem teada saada ja seda teadmist kliinilises praktikas kasutada. Stressimudelina kasutasime elektrišokki jalgadele (0.6 mA, 3 min) ja analgeesia tasemeid enne ja pärast stressi mõõtsime tail flick meetodiga. Leidsime, et CCK2–/– hiirtel tekkis samale stressile vastuseks väiksem analgeesia. See lubab oletada, et CCK2 signaali elimineerimine häirib mõningaid endogeense analgeesia mehhanisme. Järgmiseks manustasime loomadele opioidiretseptorite antagonisti naloksooni (0.01…10 mg/kg) ja kannabinoidi CB1 retseptori antagonisti rimonabanti (0.1…3 mg/kg) ja uurisime nende toimet stress-analgeesiale. Naloksoon pärssis analgeesiat mõlemal genotüübil, nii CCK2–/– kui ka metsiktüüpi hiirtel. Erinevalt naloksoonist pärssis rimonabant stress-analgeesiat ainult metsiktüüpi loomadel ja ei mõjutanud knockout hiirte vastust. See tulemus sobib hästi kokku järgnevalt teostatud geeniekspressiooni analüüsiga, milles leiti metsiktüüpi hiirtel vastuseks stressile ulatuslik kannabinoidisüsteemi aktivatsioon. See stressiga seotud aktivatsioon suuresti puudus mutantsetel loomadel. Kokkuvõtteks, CCK toime läbi CCK2 retseptorite on vajalik stressiga seotud endokannabinoidide aktivatsiooni jaoks, sest CCK2–/– hiirtel puudub stress-analgeesia kannabinoidne komponent.
Viimaseks analüüsisime ülalmainitud Affymetrix'i geeniekspressiooni kiibi neuropaatiliste loomade andmeid teise nurga alt. Uurides ainult genotüübi efekti (st jättes närvivigastuse faktori analüüsist välja) nägime, et enamus genotüübi erinevusi (st erinevalt ekspresseerunud geene CCK2–/– hiirtel) paiknes genoomis ühel konkreetsel regioonil, moodustades 40 Mbp ala CCK2 geeni ümber. Peaaegu kõik genotüübi efektina muutunud ekspressiooniga geenid olid knockout-madala ekspressioonisuunaga. Need geenid klasterdusid genoomse asukoha, mitte aga geeni funktsiooni järgi. Me tegime kindlaks, et need geenid pärinevad 129sv vanemliinist ning need ei olnud kadunud isegi pärast paljukordset tagasiristamist C57BL/6 tausta. Sellest järeldame, et segatausta tekitatud geenipuudulikkused ei võimalda geenifunktsiooni uurida nii puhtalt nagu varasemalt arvatud ja et sellistel hiirtel kirjeldatud fenotüüp ei pruugi olla ainult ühe geeni väljalülitamise tagajärg
Editorial on Special Issue “Precision Delivery of Drugs and Imaging Agents with Peptides”
Homing peptides and cell-penetrating peptides allow for systemic targeting of diseased tissues and/or efficient intracellular delivery of payloads [...
A Second Life for MAP, a Model Amphipathic Peptide
Cell-penetrating peptides (CPP) have been shown to be efficient in the transport of cargoes into the cells, namely siRNA and DNA, proteins and peptides, and in some cases, small therapeutics. These peptides have emerged as a solution to increase drug concentrations in different tissues and various cell types, therefore having a relevant therapeutic relevance which led to clinical trials. One of them, MAP, is a model amphipathic peptide with an α-helical conformation and both hydrophilic and hydrophobic residues in opposite sides of the helix. It is composed of a mixture of alanines, leucines, and lysines (KLALKLALKALKAALKLA). The CPP MAP has the ability to translocate oligonucleotides, peptides and small proteins. However, taking advantage of its unique properties, in recent years innovative concepts were developed, such as in silico studies of modelling with receptors, coupling and repurposing drugs in the central nervous system and oncology, or involving the construction of dual-drug delivery systems using nanoparticles. In addition to designs of MAP-linked vehicles and strategies to achieve highly effective yet less toxic chemotherapy, this review will be focused on unique molecular structure and how it determines its cellular activity, and also intends to address the most recent and frankly motivating issues for the future
Gene expression profiling reveals upregulation of Tlr4 receptors in Cckb receptor deficient mice
The cholecystokinin B (2) receptor knockout (Cckbr KO) protects against allodynia induced by chronic constriction injury (CCI). The mechanism of this phenomenon is unknown, but must involve persistent changes in pain modulation and/or inflammatory pathways. We performed a gene expression study in two brain areas (midbrain and medulla) after surgical induction of CCI in Cckbr KO and wild-type (wt) control mice. The patterns of gene expression differences suggest that the immune system is activated in higher brain structures following CCI in the wt mice. The strongest differences include genes related to the MAPK pathway activation and cytokine production. In Cckbr KO mice this expressional pattern was absent. In addition, we found significant elevation of the Toll-like receptor 4 (Tlr4) in the supraspinal structures of the mice with deleted Cckbr compared to wt control mice. This up-regulation is most likely induced by the deletion of Cckbr. We suggest that there is a functional deficiency in the Tlr4 pathway which disables the development of neuropathic pain in Cckbr KO mice. Indeed, real time PCR analysis detected a CCI-induced upregulation of Tlr4 and Il1b expression in the lumbar region of wt but not Cckbr KO mice. Gene expression profiling indicates that elements of the immune response are not activated in Cckbr KO mice following CCI. Our findings suggest that there may be a role for CCK in the regulation of innate immunity. © 2007 Elsevier B.V. All rights reserved
Cell-Penetrating Peptide and siRNA-Mediated Therapeutic Effects on Endometriosis and Cancer In Vitro Models
Gene therapy is a powerful tool for the development of new treatment strategies for various conditions, by aiming to transport biologically active nucleic acids into diseased cells. To achieve that goal, we used highly potential delivery vectors, cell-penetrating peptides (CPPs), as oligonucleotide carriers for the development of a therapeutic approach for endometriosis and cancer. Despite marked differences, both of these conditions still exhibit similarities, like excessive, uncoordinated, and autonomous cellular proliferation and invasion, accompanied by overlapping gene expression patterns. Thus, in the current study, we investigated the therapeutic effects of CPP and siRNA nanoparticles using in vitro models of benign endometriosis and malignant glioblastoma. We demonstrated that CPPs PepFect6 and NickFect70 are highly effective in transfecting cell lines, primary cell cultures, and three-dimensional spheroids. CPP nanoparticles are capable of inducing siRNA-specific knockdown of therapeutic genes, ribonucleotide reductase subunit M2 (RRM2), and vascular endothelial growth factor (VEGF), which results in the reduction of in vitro cellular proliferation, invasion, and migration. In addition, we proved that it is possible to achieve synergistic suppression of endometriosis cellular proliferation and invasion by combining gene therapy and hormonal treatment approaches by co-administering CPP/siRNA nanoparticles together with the endometriosis-drug danazol. We suggest a novel target, RRM2, for endometriosis therapy and as a proof-of-concept, we propose a CPP-mediated gene therapy approach for endometriosis and cancer
Modification of the linker amino acid in the cell-penetrating peptide NickFect55 leads to enhanced pDNA transfection for in vivo applications
Despite numerous efforts over the last three decades, nucleic acid-based therapeutics still lack delivery platforms in the clinical stage. Cell-penetrating peptides (CPPs) may offer solutions as potential delivery vectors. We have previously shown that designing a “kinked” structure in the peptide backbone resulted in a CPP with efficient in vitro transfection properties. Further optimization of the charge distribution in the C-terminal part of the peptide led to potent in vivo activity with the resultant CPP NickFect55 (NF55). Currently, the impact of the linker amino acid was further investigated in the CPP NF55, with the aim to discover potential transfection reagents for in vivo application. Taking into account the expression of the delivered reporter in the lung tissue of mice, and the cell transfection in the human lung adenocarcinoma cell line, the new peptides NF55-Dap and NF55-Dab* have a high potential for delivering nucleic acid-based therapeutics to treat lung associated diseases, such as adenocarcinoma.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)19/20907-72022/3056-
Formulation of Stable and Homogeneous Cell-Penetrating Peptide NF55 Nanoparticles for Efficient Gene Delivery In Vivo
Although advances in genomics and experimental gene therapy have opened new possibilities for treating otherwise incurable diseases, the transduction of nucleic acids into the cells and delivery in vivo remain challenging. The high molecular weight and anionic nature of nucleic acids require their packing into nanoparticles for the delivery. The efficacy of nanoparticle drugs necessitates the high bioactivity of constituents, but their distribution in organisms is mostly governed by the physical properties of nanoparticles, and therefore, generation of stable particles with strictly defined characteristics is highly essential. Using previously designed efficient cell-penetrating peptide NF55, we searched for strategies enabling control over the nanoparticle formation and properties to further improve transfection efficacy. The size of the NF55/pDNA nanoparticles correlates with the concentration of its constituents at the beginning of assembly, but characteristics of nanoparticles measured by DLS do not reliably predict the applicability of particles in in vivo studies. We introduce a new formulation approach called cryo-concentration, where we acquired stable and homogeneous nanoparticles for administration in vivo. The cryo-concentrated NF55/pDNA nanoparticles exhibit several advantages over standard formulation: They have long shelf-life and do not aggregate after reconstitution, have excellent stability against enzymatic degradation, and show significantly higher bioactivity in vivo