Plasmodium falciparum Cysteine-Rich Protective Antigen (CyRPA) Elicits Detectable Levels of Invasion-Inhibitory Antibodies during Natural Infection in Humans.

Plasmodium falciparum cysteine-rich protective antigen (CyRPA) is a conserved component of an essential erythrocyte invasion complex (RH5/Ripr/CyRPA) and a target of potent cross-strain parasite-neutralizing antibodies. While naturally acquired human RH5 antibodies have been functionally characterized, there are no similar reports on CyRPA. Thus, we analyzed the parasite-neutralizing activity of naturally acquired human CyRPA antibodies. In this regard, CyRPA human antibodies were measured and purified from malaria-infected plasma obtained from patients in central India and analyzed for their parasite neutralizing activity via in vitro growth inhibition assays (GIA). We report that, despite being susceptible to antibodies, CyRPA is a highly conserved antigen that does not appear to be under substantial immune selection pressure, as a very low acquisition rate for anti-CyRPA antibodies was reported in malaria-exposed Indians. We demonstrate for the first time that the small amounts of natural CyRPA antibodies exhibited functional parasite-neutralizing activity and that a CyRPA-based vaccine formulation induces highly potent antibodies in rabbits. Importantly, the vaccine-induced CyRPA antibodies exhibited a robust 50% inhibitory concentration (IC50) of 21.96 µg/ml, which is comparable to the IC50 of antibodies against the leading blood-stage vaccine candidate, reticulocyte-binding-like homologous protein 5 (RH5). Our data support CyRPA as a unique vaccine target that is highly susceptible to immune attack but is highly conserved compared to other leading candidates such as MSP-1 and AMA-1, further substantiating its promise as a leading blood-stage vaccine candidate.

Antibody Combinations Targeting the Essential Antigens CyRPA, RH5, and MSP-119 Potently Neutralize Plasmodium falciparum Clinical Isolates From India and Africa.

BACKGROUND: Targeting multiple key antigens that mediate distinct Plasmodium falciparum erythrocyte invasion pathways is an attractive approach for the development of blood-stage malaria vaccines. However, the challenge is to identify antigen cocktails that elicit potent strain-transcending parasite-neutralizing antibodies efficacious at low immunoglobulin G concentrations feasible to achieve through vaccination. Previous reports have screened inhibitory antibodies primarily against well adapted laboratory parasite clones. However, validation of the parasite-neutralizing efficacy against clinical isolates with minimal in vitro cultivation is equally significant to better ascertain their prospective in vivo potency. METHODS: We evaluated the parasite-neutralizing activity of different antibodies individually and in combinations against laboratory adapted clones and clinical isolates. Clinical isolates were collected from Central India and Mozambique, Africa, and characterized for their invasion properties and genetic diversity of invasion ligands. RESULTS: In our portfolio, we evaluated 25 triple antibody combinations and identified the MSP-Fu+CyRPA+RH5 antibody combination to elicit maximal parasite neutralization against P. falciparum clinical isolates with variable properties that underwent minimal in vitro cultivation. CONCLUSIONS: The MSP-Fu+CyRPA+RH5 combination exhibited highly robust parasite neutralization against P. falciparum clones and clinical isolates, thus substantiating them as promising candidate antigens and establishing a proof of principle for the development of a combinatorial P. falciparum blood-stage malaria vaccine.

Delivery of a Cancer-Testis Antigen-Derived Peptide Using Conformationally Restricted Dipeptide-Based Self-Assembled Nanotubes.

Use of tumor-associated antigens for cancer immunotherapy is limited due to their poor in vivo stability and low cellular uptake. Delivery of antigenic peptides using synthetic polymer-based nanostructures has been actively pursued but with limited success. Peptide-based nanostructures hold much promise as delivery vehicles due to their easy design and synthesis and inherent biocompatibility. Here, we report self-assembly of a dipeptide containing a non-natural amino acid, α,ß-dehydrophenylalanine (ΔF), into nanotubes, which efficiently entrapped a MAGE-3-derived peptide (M3). M3 entrapped in F-ΔF nanotubes was more stable to a nonspecific protease treatment and both F-ΔF and F-ΔF-M3 showed no cellular toxicity for four cancerous and noncancerous cell lines used. F-ΔF-M3 showed significantly higher cellular uptake in RAW 267.4 macrophage cells compared to M3 alone and also induced in vitro maturation of dendritic cells (DCs). Immunization of mice with F-ΔF-M3 selected a higher number of IFN-γ secreting CD8+ T cells and CD4+ T compared to M3 alone. On day 21, a tumor growth inhibition ratio (TGI, %) of 41% was observed in a murine melanoma model. These results indicate that F-ΔF nanotubes are highly biocompatible, efficiently delivered M3 to generate cytotoxic T lymphocytes responses, and able to protect M3 from degradation under in vivo conditions. The F-ΔF dipeptide-based nanotubes may be considered as a good platform for further development as delivery agents.

Comparative evaluation of the aqueous humor proteome of primary angle closure and primary open angle glaucomas and age-related cataract eyes.

PURPOSE: To analyze and compare the total proteome of aqueous humor (AH) from patients having primary angle closure glaucoma (PACG), primary open angle glaucoma (POAG) and age-related cataract. MATERIALS AND METHODOLOGY: Aqueous humor was collected from age-matched PACG, POAG and cataract patients who underwent surgery, and it was immediately stored at - 80 °C until analysis. From each sample, 25 µg of total protein was subjected to trypsin digestion and subsequently LC-MS/MS analysis was performed for the deep proteome analysis. The data acquired after the LC-MS/MS analysis were analyzed using Proteome Discoverer 1.4. The identified peptide matches were validated using percolator, at less than 1% false discovery rates. RESULTS: A total of 625, 594 and 636 proteins were identified in PACG, POAG and cataract groups, respectively (n = 9 in each group). The inter-group comparison among all these groups showed that 246 proteins were identified in all the three groups. An average of 236 ± 42, 218 ± 40 and 214 ± 62 proteins from each AH sample of PACG, POAG and cataract, respectively, was identified. There were 53 proteins commonly found in all 9 PACG AH, 59 proteins in POAG AH and 42 proteins in 9 cataracts AH samples. In the individual analysis, there were 28 proteins found in all the samples analyzed representing the "constitutive AH proteome." Spectral counting analysis of 246 proteins identified in all three group types showed significant differences in protein abundance. In proteins unique to PACG AH, 7 proteins viz. ARHGEF12, APC2, WAS, PIK3CG, ITGB1, MSN and PFN1 out of 226 were found in "Regulation of Actin Cytoskeleton" pathway, whereas in POAG 5 out of 206 proteins viz. ADCY2, ITPR1, MAPK3, MAP3K2 and TUBB1 were found in "Gap Junction" pathway. CONCLUSIONS: A qualitative as well as a quantitative comparison of proteomes of AH from PACG, POAG and age-related cataract eyes showed significant differences, thus providing clues to the disease pathophysiology.

Arginine-α, ß-dehydrophenylalanine Dipeptide Nanoparticles for pH-Responsive Drug Delivery.

PURPOSE: Nanoparticles (NPs) exhibiting responsiveness towards pH variations in organs, tissue microenvironments and cellular compartments can significantly add on to the drug delivery potential. Here, we have developed NPs from an amphipathic dipeptide, Arginine-α, ß-dehydrophenylalanine (RΔF), and tried to explore their pH responsive drug delivery potential in various cancer cells. METHODS: RΔF-NPs were architectured by harnessing the process of molecular self-assembly followed by the assessment of effect of pH on NPs morphology using zetasizer, SEM and CD. FTIR and PXRD analysis of the dipeptide and doxorubicin (Dox) were carried out for compatibility assessment followed by encapsulation of Dox in RΔF-NPs. RΔF-Dox-NPs were evaluated for pH dependent release as well as for in-vitro cellular internalization and efficacy in cancer cells. RESULTS: RΔF self-assembled to form monodispersed particles at pH 7. SEM analysis revealed a loss of overall particle morphology along with particle aggregation at highly acidic and basic pH respectively. The NPs demonstrated a slow and sustained release behaviour at pH 7 (97.64 ± 4.71% after 36 h) in comparison to pH 2 (90.27 ± 1.45% after 8 h) and pH 10 (96.39 ± 3.87% after 12 h). In-vitro efficacy studies carried-out in various cancer cells revealed that RΔF-Dox-NPs exhibited higher efficacy with 1.65, 1.95 and 13.34 fold lower IC50 values in comparison to Dox in C6, HCT-116 and AGS cell lines. CONCLUSIONS: RΔF-Dox-NPs with higher drug release at acidic pH, enhanced internalization in cancer cells along with higher cytotoxic potential can act as effective pH responsive drug delivery systems.

Efficacy of Dipeptide-Coated Magnetic Nanoparticles in Lung Cancer Models Under Pulsed Electromagnetic Field.

Lung cancer is the leading cause of cancer deaths and the overall 5-year survival rate is less than 17%. Hyperthermia is an alternative approach for the treatment of lung cancer and is associated with fewer side effects. We employed ironoxide nanoparticles in inducing localized hyperthermia in lung cancer cells using a pulsed electromagnetic field (PEMF). We synthesized, characterized and determined the uptake of dipeptide-coated iron oxide nanoparticles. Further, their ability in inducing localized hyperthermia in PEMF on lung cancer cells was assessed. Results showed nanoparticles are non-cytotoxic and showed enhanced cellular uptake in lung cancer cells. In vivo studies in nude mice lung tumor xenografts confirmed the presence in the tumors. Lung cancer cells pretreated with dipeptide-coated magnetic nanoparticles upon PEMF exposure induced cell death.

Short peptide based nanotubes capable of effective curcumin delivery for treating drug resistant malaria.

BACKGROUND: Curcumin (Ccm) has shown immense potential as an antimalarial agent; however its low solubility and less bioavailability attenuate the in vivo efficacy of this potent compound. In order to increase Ccm's bioavailability, a number of organic/inorganic polymer based nanoparticles have been investigated. However, most of the present day nano based delivery systems pose a conundrum with respect to their complex synthesis procedures, poor in vivo stability and toxicity issues. Peptides due to their high biocompatibility could act as excellent materials for the synthesis of nanoparticulate drug delivery systems. Here, we have investigated dehydrophenylalanine (ΔPhe) di-peptide based self-assembled nanoparticles for the efficient delivery of Ccm as an antimalarial agent. The self-assembly and curcumin loading capacity of different ΔPhe dipeptides, phenylalanine-α,ß-dehydrophenylalanine (FΔF), arginine-α,ß-dehydrophenylalanine (RΔF), valine-α,ß-dehydrophenylalanine (VΔF) and methonine-α,ß-dehydrophenylalanine (MΔF) were investigated for achieving enhanced and effective delivery of the compound for potential anti-malarial therapy. RESULTS: FΔF, RΔF, VΔF and MΔF peptides formed different types of nanoparticles like nanotubes and nanovesicles under similar assembling conditions. Out of these, F∆F nanotubes showed maximum curcumin loading capacity of almost 68 % W/W. Ccm loaded F∆F nanotubes (Ccm-F∆F) showed comparatively higher (IC50, 3.0 µM) inhibition of Plasmodium falciparum (Indo strain) as compared to free Ccm (IC50, 13 µM). Ccm-F∆F nano formulation further demonstrated higher inhibition of parasite growth in malaria infected mice as compared to free Ccm. The dipeptide nanoparticles were highly biocompatible and didn't show any toxic effect on mammalian cell lines and normal blood cells. CONCLUSION: This work provides a proof of principle of using highly biocompatible short peptide based nanoparticles for entrapment and in vivo delivery of Ccm leading to an enhancement in its efficacy as an antimalarial agent.

Plasmodium falciparum merozoite surface protein 3: oligomerization, self-assembly, and heme complex formation.

Merozoite surface protein 3 of Plasmodium falciparum, a 40-kDa protein that also binds heme, has been biophysically characterized for its tendency to form highly elongated oligomers. This study aims to systematically analyze the regions in MSP3 sequence involved in oligomerization and correlate its aggregation tendency with its high affinity for binding with heme. Through size exclusion chromatography, dynamic light scattering, and transmission electron microscopy, we have found that MSP3, previously known to form elongated oligomers, actually forms self-assembled filamentous structures that possess amyloid-like characteristics. By expressing different regions of MSP3, we observed that the previously described leucine zipper region at the C terminus of MSP3 may not be the only structural element responsible for oligomerization and that other peptide segments like MSP3(192-196) (YILGW) may also be required. MSP3 aggregates on incubation were transformed to long unbranched amyloid fibrils. Using immunostaining methods, we found that 5-15-µm-long fibrillar structures stained by anti-MSP3 antibodies were attached to the merozoite surface and also associated with erythrocyte membrane. We also found MSP3 to bind several molecules of heme by UV spectrophotometry, HPLC, and electrophoresis. This study suggested that its ability to bind heme is somehow related to its inherent characteristics to form oligomers. Moreover, heme interaction with a surface protein like MSP3, which does not participate in hemozoin formation, may suggest a protective role against the heme released from unprocessed hemoglobin released after schizont egress. These studies point to the other roles that MSP3 may play during the blood stages of the parasite, in addition to be an important vaccine candidate.

Rationally designed transmembrane peptide mimics of the multidrug transporter protein Cdr1 act as antagonists to selectively block drug efflux and chemosensitize azole-resistant clinical isolates of Candida albicans.

Drug-resistant pathogenic fungi use several families of membrane-embedded transporters to efflux antifungal drugs from the cells. The efflux pump Cdr1 (Candida drug resistance 1) belongs to the ATP-binding cassette (ABC) superfamily of transporters. Cdr1 is one of the most predominant mechanisms of multidrug resistance in azole-resistant (AR) clinical isolates of Candida albicans. Blocking drug efflux represents an attractive approach to combat the multidrug resistance of this opportunistic human pathogen. In this study, we rationally designed and synthesized transmembrane peptide mimics (TMPMs) of Cdr1 protein (Cdr1p) that correspond to each of the 12 transmembrane helices (TMHs) of the two transmembrane domains of the protein to target the primary structure of the Cdr1p. Several FITC-tagged TMPMs specifically bound to Cdr1p and blocked the efflux of entrapped fluorescent dyes from the AR (Gu5) isolate. These TMPMs did not affect the efflux of entrapped fluorescent dye from cells expressing the Cdr1p homologue Cdr2p or from cells expressing a non-ABC transporter Mdr1p. Notably, the time correlation of single photon counting fluorescence measurements confirmed the specific interaction of FITC-tagged TMPMs with their respective TMH. By using mutant variants of Cdr1p, we show that these TMPM antagonists contain the structural information necessary to target their respective TMHs of Cdr1p and specific binding sites that mediate the interactions between the mimics and its respective helix. Additionally, TMPMs that were devoid of any demonstrable hemolytic, cytotoxic, and antifungal activities chemosensitize AR clinical isolates and demonstrate synergy with drugs that further improved the therapeutic potential of fluconazole in vivo.

Mechanism of action of novel synthetic dodecapeptides against Candida albicans.

BACKGROUND: Three de novo designed low molecular weight cationic peptides (IJ2, IJ3 and IJ4) containing an unnatural amino acid α,ß-didehydrophenylalanine (∆Phe) exhibited potent antifungal activity against fluconazole (FLC) sensitive and resistant clinical isolates of Candida albicans as well as non-albicans and other yeast and filamentous pathogenic fungi. In the present study, their synthesis, susceptibility of different fungi and the mechanism of anti-candidal action have been elucidated. METHODS: The antimicrobial peptides (AMPs) were synthesized by solid-phase method and checked for antifungal activity against different yeasts and fungi by broth microdilution method. Anti-candidal mode of action of the peptides was investigated through detecting membrane permeabilization by confocal microscopy, Reactive Oxygen Species (ROS) generation by fluorometry, apoptosis and necrosis by flow cytometry and cell wall damage using Scanning and Transmission Electron Microscopy. RESULTS AND CONCLUSIONS: The MIC of the peptides against C. albicans and other yeast and filamentous fungal pathogens ranged between 3.91 and 250µM. All three peptides exhibited effect on multiple targets in C. albicans including disruption of cell wall structures, compromised cell membrane permeability leading to their enhanced entry into the cells, accumulation of ROS and induction of apoptosis. The peptides also showed synergistic effect when used in combination with fluconazole (FLC) and caspofungin (CAS) against C. albicans. GENERAL SIGNIFICANCE: The study suggests that the AMPs alone or in combination with conventional antifungals hold promise for the control of fungal pathogens, and need to be further explored for treatment of fungal infections.

Neoantigens and cancer-testis antigens as promising vaccine candidates for triple-negative breast cancer: Delivery strategies and clinical trials.

Immunotherapy is gaining prominence as a promising strategy for treating triple-negative breast cancer (TNBC). Neoantigens (neoAgs) and cancer-testis antigens (CTAs) are tumor-specific targets originating from somatic mutations and epigenetic changes in cancer cells. These antigens hold great promise for personalized cancer vaccines, as supported by preclinical and early clinical evidence in TNBC. This review delves into the potential of neoAgs and CTAs as vaccine candidates, emphasizing diverse strategies and delivery approaches. It also highlights the current status of vaccination modalities undergoing clinical trials in TNBC therapy. A comprehensive understanding of neoAgs, CTAs, vaccination strategies, and innovative delivery methods is crucial for optimizing neoAg-based immunotherapies in clinical practice.

Bio-piezoelectric phenylalanine-αß-dehydrophenylalanine nanotubes as potential modalities for combinatorial electrochemotherapy in glioma cells.

Bio-piezoelectric materials are endowed with characteristic features such as non-invasiveness, small energy attenuation and deep tissue penetrability. Thus, they have the ability to serve as both diagnostic and therapeutic modalities for targeting and treating various dreaded disorders scourging mankind. Herein, piezoelectric nanotubes derived from a modified amino acid-containing dipeptide, phenylalanine-αß-dehydrophenylalanine (Phe-ΔPhe; FΔF), possessing acoustic stimulation-triggered reactive oxygen species (ROS) generating ability, were employed and projected for achieving a piezo-active response enabled anti-cancer effect in glioma cells. A model anti-cancer drug doxorubicin (Dox) was also loaded into the nanotubes and the combined system depicted enhanced ROS production and cell killing under an acoustically developed piezo-catalytic environment. Cellular level assessment studies demonstrated that the dipeptide based piezoelectric nanotubes could lead to an increase in the cellular Ca2+ ion concentration, further inducing ROS-triggered cytotoxicity accompanied by high therapeutic efficacy in C6 glioma cells. Overall, our structures have the uniqueness of serving as acoustic stimulus-driven, wireless, and non-invasive electro-chemotherapeutic agents for enabling heightened cancer cell killing and may complement other chemotherapeutic modalities for treating the disease.

Anti-amyloidogenic amphipathic arginine-dehydrophenylalanine spheres capped selenium nanoparticles as potent therapeutic moieties for Alzheimer's disease.

Aggregation of both amyloid beta (Aß) peptide and hyperphosphorylated tau proteins is the major pathological hallmark of Alzheimer's disease (AD). Moieties that carry anti-amyloidogenic potency against both of the aggregating entities are considered to be promising drug candidatures for the disease. In the current work, we have synthesized amphipathic dipeptide vesicle-templated selenium nanoparticles (RΔF-SeNPs) as potential entities to combat AD. We have investigated and established their anti-amyloidogenic activity against different peptide-based amyloid models, such as the reductionist model based on the dipeptide phenylalanine-phenylalanine (FF) derived from Aß; a model based on the hexapeptide Ac-PHF6 (306VQIVYK311) derived from tau protein; and the full-length Aß42 polypeptide-based model. We also evaluated the neuroprotective characteristics of RΔF-SeNPs against FF, Ac-PHF6, and Aß42 fibril-induced toxicity in neuroblastoma, SH-SY5Y cells. RΔF-SeNPs further exhibited neuroprotective effects in streptozotocin (STZ) treated neuronal (N2a) cells carrying AD-like features. In addition, studies conducted in an intra-cerebroventricular STZ-instigated rat model of dementia revealed that RΔF-SeNP-treated animals showed improved cognitive activity and reduced Aß42 aggregate burden in brain tissues as compared with the STZ-treated group. Moreover, in vivo brain distribution studies conducted in animal models additionally demonstrated the brain-homing ability of RΔF-SeNPs. All together, these studies supported the potency of RΔF-SeNPs as efficient and propitious disease-modifying therapeutic agents for combating AD.

Detecting temporal and spatial malaria patterns from first antenatal care visits.

Pregnant women attending first antenatal care (ANC) visits represent a promising malaria surveillance target in Sub-Saharan Africa. Here we assessed the spatio-temporal relationship between malaria at ANC (n=6,471), in children at the community(n=9,362) and at health facilities (n=15,467) in southern Mozambique (2016-2019). ANC P. falciparum rates detected by quantitative polymerase chain reaction mirrored rates in children, regardless of gravidity and HIV status (Pearson correlation coefficient [PCC]>0.8, χ²<1.1), with a 2-3 months lag. Only at rapid diagnostic test detection limits at moderate-to-high transmission, multigravidae showed lower rates than children (PCC=0.61, 95%CI[-0.12-0.94]). Seroprevalence against the pregnancy-specific antigen VAR2CSA reflected declining malaria trends (PCC=0.74, 95%CI[0.24-0.77]). 80% (12/15) of hotspots detected from health facility data using a novel hotspot detector, EpiFRIenDs, were also identified with ANC data. The results show that ANC-based malaria surveillance offers contemporary information on temporal trends and the geographic distribution of malaria burden in the community.

Conformationally restricted, dipeptide-based, self-assembled nanoparticles for efficient vancomycin delivery.

Aim: Emergence of vancomycin (Van) resistance, and usage of its higher dose and short half-life are posing a serious concern. Slow and sustained release of Van using a nanodelivery system may overcome these problems. Materials & methods: Arginine-α,ß-dehydrophenylalanine (RΔF) was synthesized using solution-phase synthesis which self-assembled into nanospheres. Van was entrapped in the nanoparticles (NPs). In vitro and in vivo efficacy of Van-RΔF was determined using broth microdilution and the mouse thigh infection model, respectively. Results & conclusion: Van-RΔF NPs efficiently inhibited bacterial growth (Staphylococcus aureus), while Van alone showed limited growth inhibition in in vitro. Intravenous administration of Van-RΔF in mice with bacterial thigh infection showed enhanced efficacy (double) compared with Van alone, which indicates its high potential for further development.

Currently, microbial infections have become the most prevalent threat to human health. In the past few decades, researchers have tried different strategies to deal with these infections by developing new antimicrobial agents and/or improving the efficacy of already available antimicrobial agents. Controlled delivery of antimicrobial agents using nanosized vehicle systems has shown great promise in antimicrobial therapy. The authors have developed an ultrashort, modified, peptide-based nanoparticle system that can load vancomycin and release it in a controlled and sustained manner. Unlike other polymer-based nanoparticles, these dipeptide-based nanoparticles are easy to synthesize and highly biocompatible in nature. Vancomycin delivered via these peptide-based nanoparticles showed higher antibacterial activity than the drug alone. These results clearly indicated the high potential of this nanoformulation for further development as a delivery vehicle system for efficient antimicrobial therapy.

Process development and preclinical evaluation of a major Plasmodium falciparum blood stage vaccine candidate, Cysteine-Rich Protective Antigen (CyRPA).

Plasmodium falciparum Cysteine-Rich Protective Antigen (CyRPA) is an essential, highly conserved merozoite antigen that forms an important multi-protein complex (RH5/Ripr/CyRPA) necessary for erythrocyte invasion. CyRPA is a promising blood-stage vaccine target that has been shown to elicit potent strain-transcending parasite neutralizing antibodies. Recently, we demonstrated that naturally acquired immune anti-CyRPA antibodies are invasion-inhibitory and therefore a correlate of protection against malaria. Here, we describe a process for the large-scale production of tag-free CyRPA vaccine in E. coli and demonstrate its parasite neutralizing efficacy with commonly used adjuvants. CyRPA was purified from inclusion bodies using a one-step purification method with high purity (>90%). Biochemical and biophysical characterization showed that the purified tag-free CyRPA interacted with RH5, readily detected by a conformation-specific CyRPA monoclonal antibody and recognized by sera from malaria infected individuals thus indicating that the recombinant antigen was correctly folded and retained its native conformation. Tag-free CyRPA formulated with Freund's adjuvant elicited highly potent parasite neutralizing antibodies achieving inhibition of >90% across diverse parasite strains. Importantly, we identified tag-free CyRPA/Alhydrogel formulation as most effective in inducing a highly immunogenic antibody response that exhibited efficacious, cross-strain in vitro parasite neutralization achieving ~80% at 10 mg/ml. Further, CyRPA/Alhydrogel vaccine induced anti-parasite cytokine response in mice. In summary, our study provides a simple, scalable, cost-effective process for the production of tag-free CyRPA that in combination with human-compatible adjuvant induces efficacious humoral and cell-mediated immune response.

Chemically Modified Dipeptide Based Hydrogel Supports Three-Dimensional Growth and Functions of Primary Hepatocytes.

A huge shortage of organ donors, particularly in the case of liver, has necessitated the development of alternative therapeutic strategies. Primary hepatocytes (pHCs) transplantation has made a considerable transition from bench to bedside, but the short-term viability and functionality of pHCs in in vitro limit their use for clinical applications. Different cell culture strategies are required to maintain the proliferation of pHCs for extended periods. Here, we described the formation of a hybrid scaffold based on a modified dipeptide for the culture of pHCs. First, the dipeptide (Dp), isoleucine-α,ß-dehydrophenylalanine (IΔF) was synthesized, purified, and fully characterized. IΔF readily formed a highly stable hydrogel, which was also characterized by CD, TEM, and thioflavin T assay. The addition of soluble liver extracellular matrix (sLEM) to the dipeptide readily formed a hybrid scaffold that was characterized by TEM, and its mechanical strength was determined by rheology experiments. The hybrid scaffold was translucent, biocompatible, and proteolytically stable and, with its mechanical strength, closely mimicked that of the native liver. LEM1-Dp matrix exhibited high biocompatibility in the readily available adherent liver cell line Huh7 and primary rat hepatocyte cells (pHCs). pHCs cultured on LEM1-Dp matrix also maintained significantly higher cell viability and an escalated expression of markers related to the hepatocytes such as albumin as compared to that observed in cells cultured on collagen type I (Col I)-coated substrate plate (col-TCTP). Z-stacking of confocal laser microscopy's volume view clearly indicated pHCs seeded on top of the hydrogel matrix migrated toward the Z direction showing 3D growth. Our results indicated that low molecular weight dipeptide hydrogel along with sLEM can resemble biomimetic 3D-like microenvironments for improved pHCs proliferation, differentiation, and function. This hybrid scaffold is also easy to scale up, which makes it suitable for several downstream applications of hepatocytes, including drug development, pHCs transplantation, and liver regeneration.

Conformationally Restricted Dipeptide-Based Nanoparticles for Delivery of siRNA in Experimental Liver Cirrhosis.

Liver cirrhosis is a major health problem with multiple associated complications. The presently available drug delivery systems showed moderate site-specific delivery of antifibrotic molecules to the diseased liver; therefore, research on more effective and selective delivery systems in the context of liver cirrhosis remains a necessity in clinical investigation. The aim of the present study was to develop a peptide-based targeted nanocarrier to deliver an oligonucleotide to the hepatic sinusoidal and perivascular regions of the cirrhotic liver. We have synthesized and characterized a conformationally restricted targeted pentapeptide (RΔFRGD), which contains an unnatural amino acid, α,ß-dehydrophenylalanine (ΔF). The RΔFRGD self-assembled into spherical nanoparticles (NPs) and was characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Next, we investigated the delivery potential of the pentapeptide-based NPs to make a stable complex with a well-established small interference RNA and studied its site-specific delivery in experimental liver cirrhosis. We used siNR4A1 of the orphan nuclear receptor 4A1 (NR4A1), a well-known regulatory checkpoint for controlling liver fibrosis. Peptide NPs and their complex with siNR4A1 showed high biocompatibility against various mammalian cell lines. Hepatic tissue biodistribution analysis illustrated that targeted NPs predominantly accumulated in the cirrhotic liver compared to normal rats, specifically in sinusoidal and perivascular areas. A significant downregulation of the NR4A1 mRNA expression (-70%) andlower levels of the NR4A1/GAPDH ratio (-55%) were observed in the RΔFRGD-siNR4A1 nanocomplex-treated group in comparison to the RΔFRGD-vehicle group (RΔFRGD-Veh) at the gene and protein levels, respectively. In addition, in vivo inhibition of NR4A1 produced a significant aggravation in hepatic fibrosis compared with siRNA-vehicle-treated rats (+41% in the MT stain). The novel pentapeptide-based targeted delivery system can be further evaluated and validated for therapeutic purposes in various pathological conditions.

Rationale-based, de novo design of dehydrophenylalanine-containing antibiotic peptides and systematic modification in sequence for enhanced potency.

Increased microbial drug resistance has generated a global requirement for new anti-infective agents. As part of an effort to develop new, low-molecular-mass peptide antibiotics, we used a rationale-based minimalist approach to design short, nonhemolytic, potent, and broad-spectrum antibiotic peptides with increased serum stability. These peptides were designed to attain an amphipathic structure in helical conformations. VS1 was used as the lead compound, and its properties were compared with three series of derivates obtained by (i) N-terminal amino acid addition, (ii) systematic Trp substitution, and (iii) peptide dendrimerization. The Trp substitution approach underlined the optimized sequence of VS2 in terms of potency, faster membrane permeation, and cost-effectiveness. VS2 (a variant of VS1 with two Trp substitutions) was found to exhibit good antimicrobial activity against both the Gram-negative Escherichia coli and the Gram-positive bacterium Staphylococcus aureus. It was also found to have noncytolytic activity and the ability to permeate and depolarize the bacterial membrane. Lysis of the bacterial cell wall and inner membrane by the peptide was confirmed by transmission electron microscopy. A combination of small size, the presence of unnatural amino acids, high antimicrobial activity, insignificant hemolysis, and proteolytic resistance provides fundamental information for the de novo design of an antimicrobial peptide useful for the management of infectious disease.