Peptide Carrier Research Articles

Hollow Microparticles as a Superior Delivery System over Solid Microparticles for the Encapsulation of Peptides.

Peptides are gaining significant interests as therapeutic agents due to their high targeting specificity and potency. However, their low bioavailability and short half-lives limit their massive potential as therapeutics. The use of dense, solid particles of biodegradable polymer as a universal carrier for peptides also has its challenges, such as inefficient peptide release and low bioactivity. In this paper, it was established that hollow microparticles (h-MPs) instead of solid microparticles (s-MPs), as peptide carriers, could improve the release efficiency, while better preserving their bioactivity. Glucagon like Peptide-1 (GLP-1) was encapsulated as a model peptide. Mass loss, average molecular weight changes, intraparticle pH, polymer-peptide interaction and release studies, together with bioactivity assessment of the peptide for s-MPs and h-MPs were systematically analyzed and evaluated for efficacy. The intraparticle pH of s-MPs was as low as 2.64 whereas the pH of h-MPs was 4.99 by day 7. Consequently, 93% of the peptide extracted from h-MPs was still bioactive while only 58% of the peptide extracted from s-MPs was bioactive. Likewise, the cumulative release of GLP-1 by day 14 from h-MPs showed a cumulative amount of 88 ± 8% as compared to 33 ± 6% for s-MPs. The cumulative release of peptide can be significantly improved, and the bioactivity can be better preserved by simply using h-MPs instead of s-MPs as carriers.

The impact on the activity of acetylcholinesterase of a polylysine-ApoE peptide carrier targeting the blood brain barrier

The impact on the activity of acetylcholinesterase of a polylysine-ApoE peptide carrier targeting the blood brain barrier

The inhibition of acetylcholinesterase by a brain-targeting polylysine-ApoE peptide: biochemical and structural characterizations.

The in-trans delivery of protein therapeutics across the blood-brain barrier by K16ApoE peptide carrier has been demonstrated to improve the neurological symptoms and increase the life-span of late-infantile neuronal ceroid lipofuscinosis (LINCL) mice. However, acute toxicity of K16ApoE was observed in LINCL mice resulting in a narrow therapeutic index, limiting the potential of translating the K16ApoE into a viable drug delivery system. This study aims to unravel the toxic mechanism of action. We hypothesized that the toxic response towards the peptide was induced by inhibition of acetylcholinesterase (AChE) activity at neuro-muscular junction. Here, results from the dose-response study suggested that AChE activity was inhibited by K16ApoE at either low or high doses but not at the mid-dose where a significant increase in AChE activity was observed. Meanwhile, molecular docking simulations showed that the N-terminus of K16ApoE is capable of binding to the active site gorge of AChE. In addition to a favorable spatial orientation, this docking pose also revealed strong surface charge interactions which may account for the observed inhibitory effect. While statistical analysis of the dose response and survival ratio suggested that AChE is not the primary mechanism of action for the acute toxicity of K16ApoE, both biochemical evidence and structural analysis have assigned indirect but critical roles for AChE in the overall toxicity mechanism of this peptide carrier.

Selective Cu(I) complex with phosphine-peptide (SarGly) conjugate contra breast cancer: Synthesis, spectroscopic characterization and insight into cytotoxic action

The main disadvantage of conventional anticancer chemotherapy is the inability to deliver the correct amount of drug directly to cancer. Those molecular delivering systems are very important to destroy cancer cells selectively. Herein we report synthesis of phosphine-peptide conjugate (Ph2PCH2-Sar-Gly-OH, PSG) derived from SarGly (sarcosine-glycine), which can be easily exchanged to other peptide carriers, its oxide (OPh2PCH2-Sar-Gly-OH, OPSG) and the first copper(I) complex ([CuI(dmp)(P(Ph)2CH2-Sar-Gly-OH)], 1-PSG, where dmp stands for 2,9-dimethyl-1,10-phenanthroline). The compounds were characterized by elemental analysis, NMR (1D, 2D), UV–Vis spectroscopy and DFT (Density Functional Theory) methods. PSG and 1-PSG proved to be stable in biological medium in the presence of atmospheric oxygen for several days. The cytotoxicity of the compounds and cisplatin was tested against cancer cell lines: mouse colon carcinoma (CT26; 1-PSGIC50 = 3.12 ± 0.1), human lung adenocarcinoma (A549; 1-PSGIC50 = 2.01 ± 0.2) and human breast adenocarcinoma (MCF7; 1-PSGIC50 = 0.98 ± 0.2) as well as against primary line of human pulmonary fibroblasts (MRC-5; 1-PSGIC50 = 78.56 ± 1.1). Therapeutic index for 1-PSG (MCF7) equals 80. Intracellular accumulation of 1-PSG complex increased with time and was much higher (96%) inside MCF7 cancer cells than in normal MRC5 cells (20%). Attachment of SarGly to cytotoxic copper(I) complex via phosphine motif improved selectivity of copper(I) complex 1-PSG into the cancer cells. Precise mechanistic study revealed that the 1-PSG complex causes apoptotic cells MCF7 death with simultaneous decrease of mitochondrial membrane potential and increase of caspase-9 and -3 activities. Additionally, 1-PSG generated high level of reactive oxygen species that was the reason for oxidative damages to the sugar–phosphate backbone of plasmid DNA.

Combination of PLGA nanoparticles with mucoadhesive guar-gum films for buccal delivery of antihypertensive peptide

Oral administration of proteins and peptides still is a challenging task to overcome due to low permeability through absorptive epithelia, degradation and metabolism that lead to poor bioavailability. Attempting to overcome such limitations, an antihypertensive peptide derived from whey protein, with KGYGGVSLPEW sequence, was incorporated for the first time into polymeric nanoparticles. An experimental design was followed in order to optimize drug-loading, association efficiency, mean particle size, zeta-potential and polydispersity index of a formulation of poly(lactic-co-glycolic acid) (PLGA) nanoparticles as carriers for bioactive peptides. In sequence, peptide-loaded PLGA nanoparticles were incorporated in a guar-gum film matrix, resulting in a combined delivery system aiming to promote slow release and permeation across buccal epithelium. Neither PLGA nanoparticles, guar-gum films nor the conjugation of PLGA nanoparticles and guar-gum films (GfNp) significantly compromised in vitro TR146 human buccal carcinoma cell line viability after 12 h contact, as assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide reduction assay (MTT). In vitro release assay for developed formulations allowed to conclude that the combination of orodispersible film and nanoparticles granted a slower release of AhP when compared with PLGA or guar-gum films alone or with control. GfNp offered more effective, synergistic, in vitro permeation of TR146 cell multilayer in comparison with guar-gum films or PLGA nanoparticles alone. The combination of PLGA nanoparticles with guar-gum films represent a suitable alternative to conventional per os delivery systems, leading to an increased buccal permeability of carried antihypertensive peptide.

Insights into cell penetrating peptide conjugated gold nanoparticles for internalization into bacterial cells

Gold nanoparticles (AuNPs) functionalized with different biomolecules find extensive application in therapy, clinical diagnosis and biomedical imaging. Herein, two derivatives of TAT peptide with sequences YGRKKRRQRRR and YGRKKRRQRRR-(β-ala)3-Cys-amide were conjugated with tannic acid capped gold nanoparticles which acted as a carrier for cell penetrating peptides (CPPs) into the bacterial cells. The interaction of YGRKKRRQRRR peptide with AuNPs was non-covalent in nature whereas YGRKKRRQRRR-(β-ala)3-Cys-amide interacted covalently with the AuNPs due to presence of thiol group in cysteine which bind strongly to gold nanoparticles surface. Further, tannic acid functionalised AuNPs conjugated CPPs constructs were duly characterized using critical flocculation essay test, UV–visible and TEM. FITC was tagged over AuNPs-CPPs in order to study the intracellular distribution using confocal microscopy. The confocal results revealed that nanoconjugates (AuNP-CPPs) of 5 nm diameter exhibited strong fluorescent signal in Gram positive and Gram negative bacterial strains. The present method can also be used for the killing of bacterial cells using photo-thermal therapy and therefore can be highly useful for targeting multi-drug resistant bacteria.

Gossypol reduced the intestinal amino acid absorption capacity of young grass carp (Ctenopharyngodon idella)

This study was conducted to evaluate the effects of gossypol on the growth, intestinal histology, intestinal amino acid (AA) absorption capacity and the safe upper limit of gossypol in the diets for young grass carp (Ctenopharyngodon idella) growth. The fish were fed six diets containing different levels of free gossypol (0, 125 (121.38), 250 (243.94), 375 (363.89), 775 (759.93) and 1175 (1162.06) mg kg−1 diet) from gossypol-acetic acid for 60 days. Results indicated that gossypol could reduce the growth performance and damage the intestines of young grass carp. Interestingly, gossypol modulated the absorptive abilities of various AAs, and mechanisms are not unified in fish. Firstly, gossypol decreased the contents of tryptophan (Trp), threonine (Thr), valine (Val), phenylalanine (Phe), glycine (Gly), serine (Ser) and tyrosine (Tyr) in the three intestinal segments of fish associated with down-regulating the mRNA levels of solute carrier (SLC) 6A19b, SLC7A5, SLC7A8, SLC1A5, SLC38A2 and peptide transporter 1 (PepT1) in the intestine. Secondly, gossypol reduced the contents of methionine (Met), isoleucine (Ile), proline (Pro), cysteine (Cys) and arginine (Arg) in the proximal intestine (PI) and mid intestine (MI) of fish by down-regulating the mRNA levels of SLC7A7, SLC7A6, SLC7A1, SLC6A19b, SLC7A5, SLC7A8, SLC1A5, SLC38A2 and PepT1 in the PI and MI. Thirdly, gossypol inhibited the aspartic acid (Asp) and taurine (Tau) contents in the PI and MI of fish by down-regulating the mRNA levels of SLC1A2a, SLC1A3, PepT1 and SLC6A6 in the PI and MI. Fourthly, gossypol suppressed the lysine(Lys) and histidine (His) contents in the MI and distal intestine (DI) of fish due to down-regulating the mRNA levels of SLC7A7, SLC7A5 and SLC38A2 in the MI, and SLC7A6, SLC7A1, SLC7A8 and PepT1 in the MI and DI. Gossypol down-regulated the above AA and peptide transporters in the intestine of fish partly related to suppressing the target of rapamycin (TOR) signalling pathway. Based on percent weight gain (PWG) and feed efficiency (FE), the safe upper limit of gossypol in the diets for young grass carp growth were estimated to be 182 and 179 mg kg−1 diet, respectively. In summary, gossypol reduced the intestinal AA absorption capacity of young grass carp partly relate to damaging the intestinal structure, down-regulating the mRNA levels of AA and peptide transporters, and inhibiting the TOR signalling pathway.

Solid-in-Oil Peptide Nanocarriers for Transcutaneous Cancer Vaccine Delivery against Melanoma.

Cancer vaccines represent a prophylactic or therapeutic method of suppressing cancer by activating the adaptive immune system. The immune response is initiated by the delivery of tumor antigens to antigen presenting cells (APCs). The use of peptides as vaccine antigens is advantageous, especially in the availability and productivity of pure and defined antigens. However, their limited immunogenicity remains a major drawback, and therefore, the utilization of nanocarriers as a means of delivering antigens to target cells and/or the addition of immune stimulants have been investigated as an efficient peptide-based cancer vaccine. We have developed a solid-in-oil (S/O) nanodispersion as a transcutaneous nanocarrier for hydrophilic molecules. This system has attractive features as a peptide nanocarrier for cancer vaccines, including transcutaneous targeting of professional APCs in the skin, high encapsulation efficacy of hydrophilic molecules, and capacity for coloading with a variety of immune stimulants such as adjuvants. We therefore sought to utilize the developed S/O nanodispersion for the delivery of the tyrosine-related protein 2 peptide, TRP-2180-188, as a peptide antigen against melanoma. Transcutaneous vaccination of the S/O nanodispersion coloaded with adjuvant R-848 was associated with a significant inhibition of melanoma growth and suppression of lung metastasis in tumor-bearing mice. Our findings indicate the potential of S/O nanodispersions as an endogenous peptide carrier for cancer vaccines.

Zein nanoparticle as a novel BMP6 derived peptide carrier for enhanced osteogenic differentiation of C2C12 cells

Zein nanoparticles as a carrier system for BMP6-derived peptide were prepared by liquid–liquid phase separation procedure and characterized with SEM, DLS, FTIR and thermogravimetric methods. After peptide encapsulation, nanoparticle size increased from 236.3 ± 92.2 nm to 379.4 ± 116.8 nm. The encapsulation efficiency of peptide was 72.6% and the release of peptide from Zein nanoparticles was partly sustained in trypsin containing phosphate buffered saline (pH 7.4) for up to 14 days. Peptide-loaded nanoparticles showed similar cell viability compared with blank ones. ALP activity of C2C12 cells treated with peptide-loaded nanoparticles (500 µg/mL) was evaluated 7, 14, 21 and 28 days after culture. In peptide-loaded nanoparticles, ALP activity was significantly higher (p < .05) compared with other groups at day 14. Alizarin Red S staining showed, C2C12 cells behind peptide-loaded nanoparticles had significantly (p < .05) higher calcium deposition at day 21. The results of RT-qPCR show that the BMP-6 peptide activated expression of RUNX2 as a transcription factor. In turn, RUNX2 regulates SPP1 and BGLAP gene expression, as osteogenic marker genes. The results confirm that the peptide-loaded Zein nanoparticles, as osteoinductive material, may be used to repair small area of bone defects, with low load bearing.

Cell Penetrating Peptide-Based Redox-Sensitive Vaccine Delivery System for Subcutaneous Vaccination.

In immunotherapy, induction of potent cellular immunity by vaccination is essential to treat intracellular infectious diseases and tumors. In this work, we designed a new synthetic peptide carrier, Cys-Trp-Trp-Arg8-Cys-Arg8-Cys-Arg8-Cys, for vaccine delivery by integrating a redox-responsive disulfide bond cross-linking and cell-penetrating peptide arginine octamer. The carrier peptide bound to the antigen protein ovalbumin (OVA) via electrostatic self-assembly to form peptide/OVA nanocomposites. Then, the spontaneous oxidization of the thiols of the cysteine residues induced interpeptide disulfide bond cross-linking to construct denser peptide/OVA condensates. The cell-penetrating peptides incorporated in the carrier peptide could increase antigen uptake by antigen presenting cells. After being internalized by antigen presenting cells, the antigen could be rapidly released in cytoplasm along with degradation of the disulfide bonds by intracellular glutathione, which could promote potent CD8+ T cell immunity. The cross-linked peptide/OVA condensates were used for subcutaneous vaccination. The results showed that the peptide carrier mediated potent antigen-specific immune response by significantly increasing IgG titer; splenocyte proliferation; the secretion level of cytokines INF-γ, IL-12, IL-4, and IL-10; immune memory function, and the activation and maturation of dendritic cells. From the results, the low-molecular weight vaccine-condensing peptide with definite chemical composition could be developed as a novel class of vaccine delivery systems.

Recombinant PEP-1-SOD1 improves functional recovery after neural stem cell transplantation in rats with traumatic brain injury.

The transplantation of neural stem cells (NSCs) has been demonstrated as a potential treatment strategy for traumatic brain injury (TBI). Cu, Zn-superoxide dismutase (SOD1) is an important antioxidant enzyme that detoxifies intracellular reactive oxygen species, thereby protecting cells from oxidative damage. PEP-1, a peptide carrier, is able to deliver full-length native peptides or proteins into cells. Therefore, the current study investigated the effect of the transplantation of NSCs in combination with PEP-1-SOD1 for the treatment of experimental TBI in rats. Initially, the effect of PEP-1-SOD1 on the proliferation of NSCs was evaluated by MTT assay. PEP-1-SOD1 (0.5, 2.5 and 4.5 µM) significantly increased the proliferation rates of NSCs at 24, 48 and 72 h in a dose-dependent manner. PEP-1-SOD1 also promoted the differentiation of NSCs in vitro. The in vivo experiment showed that PEP-1-SOD1 in combination with NSC transplantation significantly improved the functional recovery of rats following TBI compared with NSC transplantation alone. A significant increase in brain aquaporin-4 (AQP4) mRNA and protein expression levels was observed 4 days post-TBI in PEP-1-SOD1, NSCs and PEP-1-SOD1 + NSCs groups compared with the saline group. The PEP-1-SOD1 + NSCs group showed a further increase of AQP4 mRNA and protein expression levels compared with the NSCs and PEP-1-SOD1 groups. In conclusion, the current data suggests that PEP-1-SOD1 may promote the proliferation and differentiation of NSCs, and thereby improve the functional recovery of TBI model rats following NSCs transplantation through upregulating the expression of AQP4.

A Review on Recent Advances in Stabilizing Peptides/Proteins upon Fabrication in Hydrogels from Biodegradable Polymers.

Hydrogels evolved as an outstanding carrier material for local and controlled drug delivery that tend to overcome the shortcomings of old conventional dosage forms for small drugs (NSAIDS) and large peptides and proteins. The aqueous swellable and crosslinked polymeric network structure of hydrogels is composed of various natural, synthetic and semisynthetic biodegradable polymers. Hydrogels have remarkable properties of functionality, reversibility, sterilizability, and biocompatibility. All these dynamic properties of hydrogels have increased the interest in their use as a carrier for peptides and proteins to be released slowly in a sustained manner. Peptide and proteins are remarkable therapeutic agents in today’s world that allow the treatment of severe, chronic and life-threatening diseases, such as diabetes, rheumatoid arthritis, hepatitis. Despite few limitations, hydrogels provide fine tuning of proteins and peptides delivery with enormous impact in clinical medicine. Novels drug delivery systems composed of smart peptides and molecules have the ability to drive self-assembly and form hydrogels at physiological pH. These hydrogels are significantly important for biological and medical fields. The primary objective of this article is to review current issues concerned with the therapeutic peptides and proteins and impact of remarkable properties of hydrogels on these therapeutic agents. Different routes for pharmaceutical peptides and proteins and superiority over other drugs candidates are presented. Recent advances based on various approaches like self-assembly of peptides and small molecules to form novel hydrogels are also discussed. The article will also review the literature concerning the classification of hydrogels on a different basis, polymers used, “release mechanisms” their physical and chemical characteristics and diverse applications.

Modification of translationally controlled tumor protein-derived protein transduction domain for improved intranasal delivery of insulin

Carrier peptides, termed protein transduction domains (PTDs), serve as provide promising vehicles for intranasal delivery of macromolecular drugs. A mutant PTD derived from human translationally controlled tumor protein (TCTP-PTD 13, MIIFRALISHKK) was reported to provide enhanced intranasal delivery of insulin. In this study, we tested whether its efficiency could be further improved by replacing amino acids in TCTP-PTD 13 or changing the amino acids in the carrier peptides from the l- to the d-form. We assessed the pharmacokinetics of PTD-mediated transmucosal delivery of insulin in normal rats and the activity of insulin in alloxan-induced diabetic rats. The safety/toxicity profile of the carrier peptides was evaluated based on the release of lactate dehydrogenase (LDH) in nasal wash fluid, body weight changes, and several biochemical parameters. Pharmacokinetic and pharmacodynamic studies showed that the l-form of a double substitution A6L, I8A (MIIFRLLASHKK), designated as l-TCTP-PTD 13M2 was the most effective carrier for intranasal insulin delivery. The relative bioavailability of insulin co-administered intranasally with l-TCTP-PTD 13M2 was 37.1% of the value obtained by the subcutaneous route, which was 1.68-fold higher than for insulin co-administered with l-TCTP-PTD 13. Moreover, co-administration of insulin plus l-TCTP-PTD 13M2 reduced blood glucose levels compared to levels in diabetic rats treated with insulin plus l-TCTP-PTD 13. There was no evidence of toxicity. These results suggest that the newly designed PTD is a useful carrier peptide for the intranasal delivery of drugs or biomolecules.

Isolation and Characterization of Two Distinct Types of Unmodified Spherical Plant Sobemovirus-Like Particles for Diagnostic and Technical Uses.

Plant virus-like particles (VLPs) structurally resemble their progenitor viruses, but are noninfectious due to absence of viral nucleic acids. Since the 1980s, VLPs have been actively studied with the aim of constructing different nanomaterials, including immunologically active carriers for peptides and whole proteins and proteinaceous shells for the packaging of different ligands.The technological developments using VLPs require large amounts of purified particles. Here, we describe the laboratory process for isolation and purification of two unmodified plant VLPs, derived from two sobemoviruses, cocksfoot mottle virus (CfMV) and rice yellow mottle virus (RYMV), which is based on cultivation of recombinant Escherichia coli cells, VLP precipitation from bacterial extracts and ultracentrifugation. The suggested purification scheme allows the production of 4-45 mg of purified sobemoviral VLPs from a 1l bacterial culture, depending on the required purity level. Additionally, we provide short protocols for VLP characterization using SDS-PAGE, agarose gel electrophoresis, ultraviolet and mass spectrometry, dynamic light scattering, and electron microscopy.

Monoclonal Antibody That Recognizes Diethoxyphosphotyrosine-Modified Proteins and Peptides Independent of Surrounding Amino Acids.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are irreversibly inhibited by organophosphorus pesticides through formation of a covalent bond with the active site serine. Proteins that have no active site serine, for example albumin, are covalently modified on tyrosine and lysine. Chronic illness from pesticide exposure is not explained by inhibition of AChE and BChE. Our goal was to produce a monoclonal antibody that recognizes proteins diethoxyphosphorylated on tyrosine. Diethoxyphosphate-tyrosine adducts for 13 peptides were synthesized. The diethoxyphosphorylated (OP) peptides cross-linked to four different carrier proteins were used to immunize, boost, and screen mice. Monoclonal antibodies were produced with hybridoma technology. Monoclonal antibody depY was purified and characterized by ELISA, western blotting, Biacore, and Octet technology to determine binding affinity and binding specificity. DepY recognized diethoxyphosphotyrosine independent of the amino acid sequence around the modified tyrosine and independent of the identity of the carrier protein or peptide. It had an IC50 of 3 × 10-9 M in a competition assay with OP tubulin. Kd values measured by Biacore and OctetRED96 were 10-8 M for OP-peptides and 1 × 10-12 M for OP-proteins. The limit of detection measured on western blots hybridized with 0.14 μg/mL of depY was 0.025 μg of human albumin conjugated to YGGFL-OP. DepY was specific for diethoxyphosphotyrosine (chlorpyrifos oxon adduct) as it failed to recognize diethoxyphospholysine, phosphoserine, phosphotyrosine, phosphothreonine, dimethoxyphosphotyrosine (dichlorvos adduct), dimethoxyphosphoserine, monomethoxyphosphotyrosine (aged dichlorvos adduct), and cresylphosphoserine. In conclusion, a monoclonal antibody that specifically recognizes diethoxyphosphotyrosine adducts has been developed. The depY monoclonal antibody could be useful for identifying new biomarkers of OP exposure.

Membrane interactions of microgels as carriers of antimicrobial peptides

Microgels are interesting as potential delivery systems for antimicrobial peptides. In order to elucidate membrane interactions of such systems, we here investigate effects of microgel charge density on antimicrobial peptide loading and release, as well as consequences of this for membrane interactions and antimicrobial effects, using ellipsometry, circular dichroism spectroscopy, nanoparticle tracking analysis, dynamic light scattering and z-potential measurements. Anionic poly(ethyl acrylate-co-methacrylic acid) microgels were found to incorporate considerable amounts of the cationic antimicrobial peptides LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) and DPK-060 (GKHKNKGKKNGKHNGWKWWW) and to protect incorporated peptides from degradation by infection-related proteases at high microgel charge density. As a result of their net negative z-potential also at high peptide loading, neither empty nor peptide-loaded microgels adsorb at supported bacteria-mimicking membranes. Instead, membrane disruption is mediated almost exclusively by peptide release. Mirroring this, antimicrobial effects against several clinically relevant bacteria (methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, and Pseudomonas aeruginosa) were found to be promoted by factors facilitating peptide release, such as decreasing peptide length and decreasing microgel charge density. Microgels were further demonstrated to display low toxicity towards erythrocytes. Taken together, the results demonstrate some interesting opportunities for the use of microgels as delivery systems for antimicrobial peptides, but also highlight several key factors which need to be controlled for their successful use.

Non-Covalent Loading of Anti-Cancer Doxorubicin by Modularizable Peptide Self-Assemblies for a Nanoscale Drug Carrier.

We prepared nanoscale, modularizable, self-assembled peptide nanoarchitectures with diameters less of than 20 nm by combining β-sheet-forming peptides tethering a cell-penetrating peptide or a nuclear localization signal sequence. We also found that doxorubicin (Dox), an anti-cancer drug, was non-covalently accommodated by the assemblies at a ratio of one Dox molecule per ten peptides. The Dox-loaded peptide assemblies facilitated cellular uptake and subsequent nuclear localization in HeLa cells, and induced cell death even at low Dox concentrations. This peptide nanocarrier motif is a promising platform for a biocompatible drug delivery system by altering the targeting head groups of the carrier peptides.

Using siRNA-based spherical nucleic acid nanoparticle conjugates for gene regulation in psoriasis

Psoriasis is a T-cell-mediated skin disease with autoimmune nature that is generally not observed in animals, this lack of a relevant experimental animal model of psoriasis has hindered the investigation of pathogenesis of disease. Application and systemic delivery of small interfering RNAs offer many effective therapeutic advantages for gene regulation in the skin.In this study, we present an IMQ animal model of psoriasis and designed a safe fusion peptide carrier, spherical nucleic acid gold nanoparticles conjugate, to improve penetration of the siRNA into the cells and skin and their targeting ability to gene regulation. We evaluated the model of psoriasis and EGFR siRNA treatment (as spherical nucleic acid nanoparticles), phenotypically (signs of erythema, scaling, inflammation and thickening), microscopic evaluation of cell proliferation and immunohistochemically evaluation of CD3, CD4, and CD8 markers. Also, we monitored suppression of EGF&EGFR genes after treatment of A431 cells by SNA-NCs. The expression of genes was validated by qRT–PCR in human skin cells.The results showed that the SNA-NCs were stable and non-toxic. In vitro experiments indicated that EGF&EGFR siRNAs conjugated with spherical nucleic acid gold nanoparticles can significantly reduce gene expression in cells. In vivo experiments showed that the topical application of siRNAs delivered by SNA-NCs through the skin can significantly inhibit the proliferation of cells. Microscopic evaluation of mice back skin and immunohistochemistry process approved Inhibitory effect of SNA-NCs siRNA in the mouse model of psoriasis.Since the proliferation of T cells was crucial for the development of a psoriatic phenotype. These results demonstrate that topical application of SNA-NCs siRNA may improve psoriatic-like skin lesions by suppressing gene expression and functional activity of T cell production.

Arginine-rich cross-linking peptides with different SV40 nuclear localization signal content as vectors for intranuclear DNA delivery

The major barriers for intracellular DNA transportation by cationic polymers are their toxicity, poor endosomal escape and inefficient nuclear uptake. Therefore, we designed novel modular peptide-based carriers modified with SV40 nuclear localization signal (NLS). Core peptide consists of arginine, histidine and cysteine residues for DNA condensation, endosomal escape promotion and interpeptide cross-linking, respectively. We investigated three polyplexes with different NLS content (10 mol%, 50 mol% and 90 mol% of SV40 NLS) as vectors for intranuclear DNA delivery. All carriers tested were able to condense DNA, to protect it from DNAase I and were not toxic to the cells. We observed that cell cycle arrest by hydroxyurea did not affect transfection efficacy of NLS-modified carriers which we confirmed using quantitative confocal microscopy analysis. Overall, peptide carrier modified with 90 mol% of SV40 NLS provided efficient transfection and nuclear uptake in non-dividing cells. Thus, incorporation of NLS into arginine-rich cross-linking peptides is an adequate approach to the development of efficient intranuclear gene delivery vehicles.

Simple and efficient knockdown of His-tagged proteins by ternary molecules consisting of a His-tag ligand, a ubiquitin ligase ligand, and a cell-penetrating peptide

We designed and synthesized hybrid molecules for a protein knockdown method based on the recognition of a His-tag fused to a protein of interest (POI). The synthesized target protein degradation inducers contained three functional moieties: a His-tag ligand (nickel nitrilotriacetic acid [Ni-NTA]), an E3 ligand (bestatin [BS] or MV1), and a carrier peptide (Tat or nonaarginine [R9]). The designed hybrid molecules, BS-Tat-Ni-NTA, MV1-Tat-Ni-NTA, BS-R9-Ni-NTA, and MV1-R9-Ni-NTA, efficiently degraded His-tagged cellular retinoic acid binding protein 2 via the ubiquitin–proteasome system (UPS). This system will become a useful tool for research into selective protein degradation inducers that act via the UPS.